Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection

During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8⁺ T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso^–/–) mice reduced CD8⁺ T-cell function in the liver and resulted in virus persistence. Furthermore, Toso^–/– DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection.More than 500 million people worldwide suffer from chronic infections with hepatitis B or hepatitis C viruses.¹ Although both viruses are poorly cytopathic, persistence of either virus can lead to chronic liver inflammation and potentially cause liversteatosis, liver cirrhosis, end-stage liver failure or hepatocellular carcinoma. Virus-specific CD8⁺ T cells are a major determinant governing the outcome of viral hepatitis due to their antiviral activity against virus-infected hepatocytes.^{2, 3, 4, 5} However, during prolonged infection, virus-specific CD8⁺ T cells are exhausted, resulting in their loss of function and consequently virus persistence.^{1, 6} Regulators influencing CD8⁺ T-cell function during chronic virus infection still remain ill defined.Inflammatory dendritic cells (iDCs) can develop from a subset of monocytes recruited to the site of inflammation.^{7, 8} This monocyte subset is characterized by the expression of CD115⁺/Ly6C^hi/CCR2⁺.⁷ iDCs express CD11c, CD11b, and Ly6C.^{9, 10, 11} IDCs that exhibit tumor necrosis factor (TNF)-α production and inducible nitric oxide synthase (iNOS) were named TNF-α and iNOS producing DCs (Tip-DCs). iDCs contribute to the elimination of pathogens following bacterial infection.^{12, 13, 14} During infection with influenza virus, iDCs enhance CD8⁺ T-cell immunopathology, but have limited impact on viral replication.^{11, 15} According to recent observations, chronic activation of toll-like receptor 9 leads to intrahepatic myeloid-cell aggregates (iMATE).¹⁶ These aggregates, which contain iDCs, are essential for T-cell activation and therefore participate in virus control.¹⁶ Co-stimulatory signals from either direct cell contact or from cytokines in combination with continued antigen contact in iMATEs lead to proliferation and activation of virus-specific T cells.¹⁶ These observations suggest that infiltration of professional antigen-presenting cells into target organs is important for the maintenance of strong antiviral cytotoxic CD8⁺ T-cell activity. Factors regulating iDC infiltration into the liver remain poorly understood.Toso is a membrane protein whose extracellular domain has homology to the immunoglobulin variable (IgV) domains. The cytoplasmic region has partial homology to the FAST kinase (Fas-activated serine/threonine kinase).¹⁷ Toso is expressed on B cells and activated T cells¹⁷ and is overexpressed in B-cell lymphomas.^{18, 19} Expression of Toso can influence survival of macrophages.²⁰ Originally, Toso was described as an inhibitor of FAS signaling.^{17, 21} More recently, a role of Toso in IgM binding and TNFR signaling was also demonstrated^{22, 23, 24} and consistently, Toso-deficient animals are protected from lipopolysaccharide (LPS)-induced septic shock.^{24, 25} Recently, we identified a role of Toso in the activation of granulocytes, monocytes, and DCs.^{26, 27, 28} During infection with Listeria, the expression of Toso regulated granulocyte function.^{26, 27} The role of Toso in the function of monocytes and other myeloid cells still remains to be further elucidated.In this study, we investigated the role of Toso during chronic viral infection by using the murine lymphocytic choriomeningitis virus (LCMV). We report that Toso promotes the differentiation and maturation of iDCs at virus-infected sites, which were essential for effector CD8⁺ T-cell function and in accelerating the control of the virus. We further tested the role of Toso in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model and found that Toso was required to trigger diabetes in RIP-GP mice. Taken together, we have identified an essential role of Toso in the differentiation and maturation of iDCs, which is essential for the control of persistence-prone virus infection and triggering of autoimmune disease.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn²⁺ level ([Zn²⁺]_c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia–reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn²⁺]_c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn²⁺]_c but abolished the cytosolic Zn²⁺ accumulation during reperfusion as well as ROS-elicited increases in the [Zn²⁺]_c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn²⁺]_c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.Transient ischemia is a major cause of chronic neurological disabilities including memory impairment and cognitive dysfunctions in stroke survivors.^{1, 2} The underlying mechanisms are complicated and multiple, and remain not fully understood.³ It is well documented in rodents, non-human primates and humans that pyramidal neurons in the CA1 region of the hippocampus are particularly vulnerable and these neurons are demised after transient ischemia, commonly referred to as the delayed neuronal death.⁴ Studies using in vitro and in vivo models of transient ischemia have demonstrated that an increase in the [Zn²⁺]_c or cytosolic Zn²⁺ accumulation is a critical factor.^{5, 6, 7, 8, 9, 10, 11} There is evidence supporting a role for ischemia-evoked release of vesicular Zn²⁺ at glutamatergic presynaptic terminals and subsequent entry into postsynaptic neurons via GluA2-lacking AMPA subtype glutamate receptors (AMPARs) to raise the [Zn²⁺]_c.^{12, 13, 14, 15, 16} Upon reperfusion, while glutamate release returns to the preischemia level,¹⁷ Zn²⁺ can activate diverse ROS-generating machineries to generate excessive ROS as oxygen becomes available, which in turn elicits further Zn²⁺ accumulation during reperfusion.^{18, 19} ROS generation and cytosolic Zn²⁺ accumulation have a critical role in driving delayed CA1 pyramidal neuronal death,^{7, 12, 20, 21, 22} but the molecular mechanisms underlying such a vicious positive feedback during reperfusion remain poorly understood.Transient receptor potential melastatin-related 2 (TRPM2) forms non-selective cationic channels; their sensitivity to activation by ROS via a mechanism generating the channel activator ADP-ribose (ADPR) confers diverse cell types including hippocampal neurons with susceptibility to ROS-induced cell death, and thus TRPM2 acts as an important signaling molecule mediating ROS-induced adversities such as neurodegeneration.^{23, 24, 25, 26} Emergent evidence indeed supports the involvement of TRPM2 in transient ischemia-induced CA1 pyramidal neuronal death.^{27, 28, 29, 30} This has been attributed to the modulation of NMDA receptor-mediated signaling; despite that ROS-induced activation of the TRPM2 channels results in no change in the excitability of neurons from the wild-type (WT) mice, TRPM2 deficiency appeared to favor prosurvival synaptic Glu2A expression and inhibit prodeath extrasynaptic GluN2B expression.³⁰ A recent study suggests that TRPM2 activation results in extracellular Zn²⁺ influx to elevate the [Zn²⁺]_c.³¹ The present study, using TRPM2-deficient mice in conjunction with in vivo and in vitro models of transient global ischemia, provides compelling evidence to show ROS-induced TRPM2 activation during reperfusion as a crucial mechanism determining the delayed cytosolic Zn²⁺ accumulation, CA1 neuronal death and postischemic memory impairment.     

Cellular prion protein promotes post-ischemic neuronal survival,angioneurogenesis and enhances neural progenitor cell homing via proteasome inhibition

Although cellular prion protein (PrP^c) has been suggested to have physiological roles in neurogenesis and angiogenesis, the pathophysiological relevance of both processes remain unknown. To elucidate the role of PrP^c in post-ischemic brain remodeling, we herein exposed PrP^c wild type (WT), PrP^c knockout (PrP−/−) and PrP^c overexpressing (PrP+/+) mice to focal cerebral ischemia followed by up to 28 days reperfusion. Improved neurological recovery and sustained neuroprotection lasting over the observation period of 4 weeks were observed in ischemic PrP+/+ mice compared with WT mice. This observation was associated with increased neurogenesis and angiogenesis, whereas increased neurological deficits and brain injury were noted in ischemic PrP−/− mice. Proteasome activity and oxidative stress were increased in ischemic brain tissue of PrP−/− mice. Pharmacological proteasome inhibition reversed the exacerbation of brain injury induced by PrP−/−, indicating that proteasome inhibition mediates the neuroprotective effects of PrP^c. Notably, reduced proteasome activity and oxidative stress in ischemic brain tissue of PrP+/+ mice were associated with an increased abundance of hypoxia-inducible factor 1α and PACAP-38, which are known stimulants of neural progenitor cell (NPC) migration and trafficking. To elucidate effects of PrP^c on intracerebral NPC homing, we intravenously infused GFP⁺ NPCs in ischemic WT, PrP−/− and PrP+/+ mice, showing that brain accumulation of GFP⁺ NPCs was greatly reduced in PrP−/− mice, but increased in PrP+/+ animals. Our results suggest that PrP^c induces post-ischemic long-term neuroprotection, neurogenesis and angiogenesis in the ischemic brain by inhibiting proteasome activity.Endogenous neurogenesis persists in the adult rodent brain within distinct niches such as the subventricular zone (SVZ) of the lateral ventricles,^{1, 2, 3, 4} which host astrocyte-like neural stem cells and neural progenitor cells (NPCs). Focal cerebral ischemia stimulates neurogenesis, and NPCs proliferate and migrate towards the site of lesion where they eventually differentiate.^{5, 6, 7} In light of low differentiation rates and high cell death rates of new-born cells,^{6, 8, 9} post-stroke neurogenesis is scarce.¹⁰Cellular prion protein (PrP^c) is a glycoprotein that is attached to cell membranes by means of a glycosylphosphatidylinositol anchor.¹¹ Although PrP^c is ubiquitously expressed, it is most abundant within the central nervous system. Conversion into its misfolded isoform PrP^sc causes neurodegenerative diseases such as Creutzfeldt-Jacob disease.^{11, 12} While a large body of studies analyzed the role of PrP^sc in the context of transmissible spongiform encephalopathies, little is known about the physiological role of PrP^c. Studies performed during both ontogenesis and adulthood suggest that PrP^c regulates neuronal proliferation and differentiation, synaptic plasticity and angiogenesis.^{13, 14, 15, 16, 17, 18} The role of these processes under pathophysiological conditions, however, is largely unknown.Previous reports suggested a role of PrP^c in post-ischemic neuroprotection.^{19, 20, 21, 22, 23, 24} Thus, PrP^c was found to be overexpressed in ischemic brain tissue.^{19, 20, 21, 22, 23, 24} PrP^c deficiency aggravated ischemic brain injury, possibly via enhanced ERK-1/2 activation and reduced phosphorylation of Akt, thus ultimately culminating in increased caspase-3 activity,^{21, 24} whereas PrP^c overexpression protected against ischemia.^{19, 20, 21, 22, 23, 24} Nevertheless, these studies focused on acute injury processes with a maximal observation period of 3 days, leaving the biological role of PrP^c in post-stroke neurogenesis and angiogenesis unanswered. To clarify the role of PrP^c in the post-acute ischemic brain, we herein exposed PrP^c wild type (WT), PrP^c knockout (PrP−/−) and PrP^c overexpressing (PrP+/+) mice to focal cerebral ischemia induced by intraluminal middle cerebral artery (MCA) occlusion, evaluating effects of PrP^c on neurological recovery, ischemic injury, neurogenesis and angiogenesis, as well as the homing and efficacy of exogenously delivered NPCs.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

Chronic restraint stress decreases the repair potential from mesenchymal stem cells on liver injury by inhibiting TGF-β1 generation

Chronic psychological stress has been demonstrated to play an important role in several severe diseases, but whether it affects disease therapy or not remains unclear. Mesenchymal stem cells (MSCs) have been demonstrated to have therapeutic potentials in treating tissue injury based on their multidifferentiation potential toward various cell types. We investigated the effect of chronic restraint stress on therapeutic potential of MSCs on carbon tetrachloride (CCl₄)-induced liver injury in mice. CCl₄-induced mice were injected with enhanced green fluorescent protein–MSCs, which was followed by chronic restraint stress administration. Corticosterone and RU486, a glucocorticoid receptor (GR) antagonist, were employed in vivo and in vitro, too. In the present study, we illustrated that MSCs could repair liver injury by differentiating into myofibroblasts (MFs) which contribute to fibrosis, whereas stress repressed differentiation of MSCs into MFs displayed by reducing α-smooth muscle actin (α-SMA, a solid marker of MFs) expression. Whereas RU486 could maintain the liver injury reduction and liver fibrosis increases induced by MSCs in stressed mice and block the decrease of α-SMA expression induced by stress. Furthermore, chronic stress inhibited MFs differentiation from MSCs by inhibiting transforming growth factor-β1 (TGF-β1)/Smads signaling pathway which is essential for MFs differentiation. Chronic stress reduced autocrine TGF-β1 of MSCs, but not blunted activation of Smads. All these data suggested that corticosterone triggered by chronic stress impaired liver injury repair by MSCs through inhibiting TGF-β1 expression which results in reduced MFs differentiation of MSCs.Liver fibrosis is a wound repairment event in response to chronic injuries induced by a series of causes, such as viral hepatitis infection, alcohol, drugs, autoimmune reaction and metabolic diseases,¹ which is characterized by excessive deposition of extracellular matrix proteins. If liver injury could not be repaired in time, the fibrosis would continue and come to a bad cycle that will alter the balance of matrix secretion and degradation. Cirrhosis, the end stage of fibrosis, mortality rate which increases speedily worldwide,² appears to be a large health burden in the world. There is still no effective and feasible treatment of cirrhosis apart from orthotopic liver transplantation.³ Therefore, treating liver injury at early stage seems to be crucial to arrest cirrhosis progression. In general, some factors resulting in liver injury could not be removed; hence, alternative strategies to repair liver injury at early stage needs to be developed.With the growing enthusiasm of stem cell therapy, the application of mesenchymal stem cells (MSCs) on liver injury repair attracts more and more attention. In the trend of stem cell therapy, there are still unresolved problems in clinical application, such as the risk of teratoma formation, ethical issue, heterogeneity rejection and normalized production. However, MSCs become the most promising candidates for treatment in recent years because they are free of ethical concerns, without the risk of teratoma formation, and with low immunogenicity. MSCs have been isolated from a wide array of tissues successfully^{4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15} and can be cultured in vitro. Dependent on the nature of injurytropism and multipotent differentiation capacity, they have been shown to be highly effective to treat various tissue injury and degenerative diseases, such as myocardial infarction, liver cirrhosis, spinal cord injury, bone damage, cornea damage, burn-induced skin defects and other tissue injuries.¹⁶ There have been reports demonstrating that exogenous MSCs can repair damaged liver, but the mechanisms are diverse.^{17, 18}It has been reported that myofibroblasts (MFs) are activated and contribute to wound healing after tissue injury.¹⁹ Hepatic stellate cells are not the only sources of MFs,²⁰ as MSCs can differentiate into MFs too.^{21, 22} Hence, it is suggested that MSCs repair liver injury through differentiating into MFs which is consistent with our results to some extent. In our study, exogenerous MSCs in early stage of liver injury could differentiate into MFs which contribute to liver fibrosis, and repaired liver injury in carbon tetrachloride (CCl₄)-induced mouse model. Therefore, we illustrated that elevated fibrosis exerted by MSCs at early stage of liver injury could reduce liver damage, even though fibrosis at late stage of liver injury results in liver failure. Transforming growth factor-β1 (TGF-β1), as a known growth factor associated with liver fibrosis, was documented to be involved in MFs differentiation from stromal cell types by inducing the expression of α-smooth muscle actin (α-SMA), a reliable marker of differentiated MFs.^{19, 23, 24, 25, 26, 27, 28, 29, 30, 31} There has been reports demonstrating that MSCs express α-SMA after TGF-β1 treatment,^{24, 25, 32, 33} and autocrine of TGF-β1 from MSCs after TGF-β1 administration has been reported too.³³During stem cell treatment, there are various factors affecting the therapy efficiency. Despite the attention paid to their own properties of MSCs, there is little consideration on the mental status of patients. Chronic stress, as a negative emotion,³⁴ accompanies with patients and exists in the process of disease therapy. Chronic stress has an important role in the occurrence and development of various considerable diseases among cardiovascular system, digestive system, immune system and nervous system. However, the role of chronic stress in the efficiency of MSCs therapy continues to be unclear. In stress system, the hypothalamic–pituitary–adrenal and the sympathetic–adrenal–medullary axises are activated, and thereby provoke the releasing of glucocorticoid (GC) (corticosterone in rodents and cortisol in humans^{35, 36}) and adrenal hormones, which are the main stress hormones. We are eager to know whether response to psychological stress of central nervous system influences therapeutic effect of MSCs on liver injury. In our study, mice were subjected to restraint stress after MSCs injection in CCl₄-induced liver fibrosis model. Here, we demonstrated that stress repressed the function of MSCs in liver injury repair through directly affecting on MSCs.     

Augmentation of NAD+ by NQO1 attenuates cisplatin-mediated hearing impairment

Cisplatin (cis-diaminedichloroplatinum-II) is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD⁺) has emerged as a key regulator of cellular energy metabolism and homeostasis. Here, we demonstrate for the first time that, in cisplatin-mediated ototoxicity, the levels and activities of SIRT1 are suppressed by the reduction of intracellular NAD⁺ levels. We provide evidence that the decrease in SIRT1 activity and expression facilitated by increasing poly(ADP-ribose) transferase (PARP)-1 activation and microRNA-34a through p53 activation aggravates cisplatin-mediated ototoxicity. Moreover, we show that the induction of cellular NAD⁺ levels using β-lapachone (β-Lap), whose intracellular target is NQO1, prevents the toxic effects of cisplatin through the regulation of PARP-1 and SIRT1 activity. These results suggest that direct modulation of cellular NAD⁺ levels by pharmacological agents could be a promising therapeutic approach for protection from cisplatin-induced ototoxicity.Cisplatin (cis-diamminedichloroplatinum (II)) is a chemotherapeutic agent extensively used to treat a variety of solid tumors in the head and neck, bladder, lung, ovaries, testicles, and uterus.¹ However, progressive irreversible side effects of cisplatin, including nephrotoxicity and ototoxicity, greatly impair the patient''s quality of life and frequently result in the need to lower the dosage during treatment or discontinuation of the treatment. Cisplatin ototoxicity primarily occurs in the cochlea and is generally caused by apoptotic damage to the outer hair cells (OHCs), spiral ganglion cells, and the marginal cells of the stria vascularis. In recent years, studies have demonstrated that cisplatin ototoxicity is also closely related to the damage of cochlear tissue by increased production of reactive oxygen species (ROS) and accompanied by the depletion of antioxidant substances and increased lipid peroxidation.^{2, 3} ROSs, particularly the hydroxyl radical, have a critical role in cisplatin-induced p53 activation through DNA damage.⁴ Although it is not easy to differentiate the cause from the consequence, a positive feedback loop between inflammatory cytokines and oxidative stress that worsen the cochlear damage is considered as one of the major mechanisms that facilitate cisplatin-induced hearing impairment.⁵ Interestingly, p53 and NF-κB have been described as key mediators of cisplatin-induced toxicity because of their involvement in oxidative stress, DNA damage, and inflammation through a mutual feedback process of ‘cause and effect.''^{6, 7} In addition, activities of p53 and NF-κB could be regulated by post-translational modifications, including phosphorylation and acetylation. Recent studies have reported that acetylated p53 and NF-κB are correlated with cisplatin-induced toxicity. Furthermore, acetylation of p53 and NF-κB is critically involved in cisplatin-induced renal injury.^{8, 9}Cellular nicotinamide adenine dinucleotide (NAD⁺) and NADH levels have been shown to be important mediators of energy metabolism and cellular homeostasis.^{10, 11} As NAD⁺ acts as a cofactor for various enzymes, including sirtuins (SIRTs), poly(ADP-ribose) transferases (PARPs), and cyclic ADP (cADP)-ribose synthases,^{12, 13} the regulation of NAD⁺ level may have therapeutic benefits through its effect on NAD⁺-dependent enzymes. SIRTs, NAD⁺-dependent protein deacetylases, are present as seven homologs of Sir2 (SIRT1-7) that show differential subcellular localizations in mammals.¹¹ Among these, nuclear SIRT1 is activated under energy stress conditions, such as fasting, exercise, or low glucose availability. ¹⁴ SIRT1 has a key role in metabolism, development, stress response, neurogenesis, hormone responses, and apoptosis^{15, 16} by deacetylation of substrates, such as NF-κB, FOXO, p53, and histones.^{17, 18, 19}PARPs, the most abundant ADP-ribosyl transferases, also use NAD⁺ to generate large amounts of poly(ADP-ribose) (PAR), which facilitate the recruitment of DNA repair factors. In particular, PARP-1 is a DNA damage sensor that can be activated in response to DNA damage by various pathophysiological conditions, including oxidative stress and inflammatory injury. However, excessive hyperactivation of PARP-1 causes the depletion of intracellular NAD⁺ and ATP levels, which eventually leads to cell death.^{20, 21} PARP-1 activation is also known as one of the important pathogenic mechanisms in cisplatin-induced toxicity.^{22, 23}A cytosolic antioxidant flavoprotein NADH:quinone oxidoreductase 1 (NQO1) catalyzes the reduction of quinones to hydroquinones by utilizing NADH as an electron donor, which consequently increases intracellular NAD⁺ levels.^{24, 25} In addition, accumulation evidence suggests that NQO1 has a role in other biological activities, including anti-inflammatory processes, scavenging of superoxide anion radicals, and stabilization of p53 and other tumor suppressor proteins.^{26, 27, 28} Several substrates of NQO1 enzyme, including mitomycin C, RH1, AZQ, Coenzyme Q10, and idebenone, have been identified,^{29, 30} of which β-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-dione; β-Lap) is recently well studied as a substrate of NQO1.^{31, 32} β-Lap was first isolated from the bark of the lapacho tree and reported to inhibit tumor cell line growth.³³ However, recent reports indicate that the conversion of NADH to NAD⁺ by NQO1 and β-Lap has beneficial effects on several characteristics of metabolic syndrome, for example, prevention of health decline in aged mice, amelioration of obesity or hypertension, prevention of arterial restenosis, and protection against salt-induced renal injury.^{34, 35, 36, 37, 38} Furthermore, we recently have demonstrated that conversion of NADH to NAD+ by NQO1 and β-Lap suppresses cisplatin-induced acute kidney injury by downregulating potential damage mediators such as oxidative stress and inflammatory responses.⁹Although a link between NAD⁺-dependent molecular events and cellular metabolism is evident, it remains unclear whether modulation of NAD⁺ levels has an impact on cisplatin-induced hearing impairment. Therefore, herein we investigated the role of NAD⁺ metabolism on cisplatin-induced cochlear dysfunction, and the effect of increased levels of intracellular NAD⁺ facilitated by β-Lap on cisplatin-induced hearing impairment with a particular interest in NAD⁺-dependent enzymatic pathways including SIRTs and PARPs.     

Apoptotic mechanisms during competition of ribosomal protein mutant cells: roles of the initiator caspases Dronc and Dream/Strica

Heterozygosity for mutations in ribosomal protein genes frequently leads to a dominant phenotype of retarded growth and small adult bristles in Drosophila (the Minute phenotype). Cells with Minute genotypes are subject to cell competition, characterized by their selective apoptosis and removal in mosaic tissues that contain wild-type cells. Competitive apoptosis was found to depend on the pro-apoptotic reaper, grim and head involution defective genes but was independent of p53. Rp/+ cells are protected by anti-apoptotic baculovirus p35 expression but lacked the usual hallmarks of ‘undead'' cells. They lacked Dronc activity, and neither expression of dominant-negative Dronc nor dronc knockdown by dsRNA prevented competitive apoptosis, which also continued in dronc null mutant cells or in the absence of the initiator caspases dredd and dream/strica. Only simultaneous knockdown of dronc and dream/strica by dsRNA was sufficient to protect Rp/+ cells from competition. By contrast, Rp/Rp cells were also protected by baculovirus p35, but Rp/Rp death was dronc-dependent, and undead Rp/Rp cells exhibited typical dronc-dependent expression of Wingless. Independence of p53 and unusual dependence on Dream/Strica distinguish competitive cell death from noncompetitive apoptosis of Rp/Rp cells and from many other examples of cell death.In Drosophila, heterozygous mutation of many ribosomal protein gene loci leads to the dominant ‘Minute'' phenotype, named for its small thin bristles.^{1, 2} Minute animals show a dominant developmental delay. In addition, Minute (that is, Rp/+) cells tend to be lost from mosaics that contain wild-type cells, making it difficult for clones of Rp/+ genotypes to survive and contribute to the adult.^{3, 4, 5, 6, 7} Such conditional cell viability that depends on a heterotypic cellular environment is termed ‘cell competition''.⁴Competition of Rp/+ clones is suppressed by equalizing growth rates through starvation⁸ or nonmosaic mutation of a second Rp locus.⁴ Hyperplastic clones that express higher levels of myc^{9, 10} or lower levels of the Salvador-Hippo-Warts pathway tumor suppressors out-compete nearby wild-type cells, that is, they are ‘super-competitors''.^{7, 11} Competition based on c-myc also occurs in mouse embryogenesis.¹² Differential growth is not always sufficient to cause cell competition, as cells growing rapidly due to elevated CyclinD/Cdk4 activity or higher activity of the insulin/IGF pathway are not super-competitive.⁹ Differences in Jak/Stat signaling, Wg signaling and cell adhesion are also reported to generate cell competition.^{13, 14, 15} These findings suggest that cell competition arises from specific interactions between cells, rather than as a general consequence of differential growth.Apoptotic cell death is a fundamental part of cell competition. Elimination of Rp/+ clones is delayed by expression of the caspase inhibitor baculovirus p35.⁵ Apoptosis of Rp/+ cells also occurs when clones of wild-type cells arise in Rp/+ backgrounds, predominantly among Rp/+ cells nearby wild-type cells.^{6, 16} As expected, such apoptosis is prevented by expression of baculovirus p35 or DIAP1.^{6, 16, 17}Cell competition has been hypothesized to contribute to human cancer, because most tumors have an altered genotype, and because many genes implicated in cell competition are homologs of oncogenes and tumor suppressors.^{18, 19, 20, 21} Cell competition may contribute to homeostasis of organ growth^{4, 9} and to antitumor surveillance.^{22, 23, 24, 25, 26}Cell competition may be a means to eliminate certain categories of aneuploid cells.^{27, 28} Seventy-nine ribosomal protein genes, sixty-six of which are haploinsufficient Minute loci, are distributed throughout the Drosophila genome.² Copy number changes to parts of the genome are likely to perturb relative dose of Rp/+ genes, and those that reduce Rp gene dose could be subject to cell competition. This suggests cell competition can eliminate some aneuploid cells even after DNA damage responses have ceased.^{27, 28, 29}In humans, heterozygosity for multiple different Rp mutations causes Diamond Blackfan Anemia.³⁰ Accumulation of ribosomal assembly intermediates or of unassembled ribosomal proteins in these genotypes activates p53, for example through the binding of the p53 ubiquitin ligase Mdm2 by RpL11 or RpL5.³¹ The p53 pathway leads to cell cycle arrest and/or apoptosis,³² and loss of hematopoietic stem cells causes anemia. Diamond Blackfan Anemia is a condition of nonmosaic individuals, so its relationship to cell competition is unclear.The uncertain nature of the cell interactions that trigger competition might be illuminated if the initiation of competitive apoptosis was understood. The Drosophila genome encodes three potential initiator caspases that might be activated through long prodomains, and four effector caspase zymogens lacking prodomains that are activated by initiator caspases and by one another.³³ Here, the p53 and initiator caspase requirements for competitive cell death of Rp/+ cells were determined. Whereas Dronc is the initiator caspase for most apoptosis in Drosophila,^{34, 35, 36, 37} we found that competitive cell death could occur without dronc or p53. Experiments that eliminated multiple initiator caspases simultaneously demonstrated that competitive apoptosis of Rp/+ cells required Dronc and Dream/Strica redundantly, a difference from most other apoptotic genotypes in Drosophila, for example, Rp/Rp cells generated in these experiments died in a Dronc-dependent manner.     

TRPC1-mediated Ca2+ entry is essential for the regulation of hypoxia and nutrient depletion-dependent autophagy

Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca²⁺ is suggested to have an important role in cell survival, the ion channel(s) involved in autophagy have not been identified. Here we demonstrate that increase in intracellular Ca²⁺ via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca²⁺ entry via the TRPC1 channel, which was inhibited by the addition of 2APB and {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365. Importantly, TRPC1-mediated Ca²⁺ entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca²⁺ influx, decreased autophagy, and increased cell death. Furthermore, the primary salivary gland cells isolated from mice exposed to hypoxic conditions also showed increased expression of TRPC1 as well as increase in Ca²⁺ entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca²⁺ influx via TRPC1 in regulating autophagy to protect against cell death.Autophagy is a cellular process responsible for the delivery of proteins or organelles to lysosomes for its degradation. Autophagy participates not only in maintaining cellular homeostasis, but also promotes cell survival during cellular stress situations.^{1, 2} The stress conditions including nutrient starvation, hypoxia conditions, invading microbes, and tumor formation, have been shown to induce autophagy that allows cell survival in these stressful or pathological situations.¹ In addition, autophagy also recycles existing cytoplasmic components to generate the molecules that are required to sustain the most vital cellular functions.³ Till date, three forms of autophagy have been identified, which are designated as chaperone-mediated autophagy, microautophagy, and macroautophagy.⁴ Although the precise mechanism as to how autophagy is initiated is not well understood, many of the genes first identified in yeast that are involved in autophagy have orthologs in other eukaryotes including human homologs.^{5, 6} The presence of similar genes in all organisms suggests that autophagy might be a phenomenon that is evolutionally conserved that is essential for cell survival. In addition, since autophagy delivers a fresh pool of amino acids and other essential molecules to the cell, initiation of autophagy is highly beneficial particularly during nutritional stress situations or tissue remodeling during development and embryogenesis.⁶ Consequently, impaired or altered autophagy is often implicated in several pathologies, like neurodegenerative disorders and cancer,^{7, 8, 9} which again highlight its importance.Ca²⁺ has a vital role in the regulation of a large number of cellular processes such as cell proliferation, survival, migration, invasion, motility, and apoptosis.^{10, 11} To perform functions on such a broad spectrum, the cells have evolved multiple mechanisms regulating cellular Ca²⁺ levels, mainly by regulating the function of various Ca²⁺ channels present in different locations. Mitochondrial, ER, lysosomal, and cytosolic Ca²⁺ levels are regulated by Ca²⁺ permeable ion channels localized either on the membranes of the intracellular organelles or on the plasma membrane.¹⁰ The Ca²⁺ permeable channels, including families of TRPCs, Orais, voltage-gated, two-pore, mitochondrial Ca²⁺ uniporter, IP₃, and ryanodine receptors have all been identified to contribute towards changes in intracellular Ca²⁺ ([Ca²⁺]_i).^{10, 12, 13, 14} Channels of the TRPCs and Orai families have been related to several Ca²⁺-dependent physiological processes in various cell types, ranging from cell proliferation to contractility, to apoptosis under both physiological and pathological conditions.¹² Moreover, it has been suggested that intracellular Ca²⁺ is one of the key regulators of autophagy;¹⁵ however, the possible role of Ca²⁺ in autophagy is still inconclusive. Many reports also suggest that Ca²⁺ inhibits autophagy,^{16, 17, 18} whereas others have indicated a stimulatory role for Ca²⁺ towards autophagy.^{19, 20, 21} Furthermore, the identity of the major Ca²⁺ channel(s) involved in autophagy is not known. Members of the TRPC family have been suggested as mediators of Ca²⁺ entry into cells. Activation of the G-protein (G_q/11–PLC pathway) leads to the generation of second messenger IP₃.^{10, 22} IP₃ binds to the IP₃R, which initiates Ca²⁺ release from the ER stores, thereby facilitating stromal interacting molecule-1 (STIM1) to rearrange and activate Ca²⁺ entry via the store-operated channels.²² Two families of proteins (TRPCs and Orais) have been identified as potential candidates for SOC-mediated Ca²⁺ entry.^{12, 22} However, their role in autophagy has not yet been determined. Thus, here we investigated the role of Ca²⁺ entry channels (TRPCs and Orais) in autophagy and show that both hypoxia-mimetic and nutrient depression induces autophagy in two different cell lines. Furthermore, our data indicates that autophagy was dependent on TRPC1-mediated increase in intracellular Ca²⁺ levels, suggesting that TRPC1 has an important role in regulating autophagy and inhibiting cell death.     

NaCl-Induced Alternations of Cellular and Tissue Ion Fluxes in Roots of Salt-Resistant and Salt-Sensitive Poplar Species

Using the scanning ion-selective electrode technique, fluxes of H⁺, Na⁺, and Cl⁻ were investigated in roots and derived protoplasts of salt-tolerant Populus euphratica and salt-sensitive Populus popularis 35-44 (P. popularis). Compared to P. popularis, P. euphratica roots exhibited a higher capacity to extrude Na⁺ after a short-term exposure to 50 mm NaCl (24 h) and a long term in a saline environment of 100 mm NaCl (15 d). Root protoplasts, isolated from the long-term-stressed P. euphratica roots, had an enhanced Na⁺ efflux and a correspondingly increased H⁺ influx, especially at an acidic pH of 5.5. However, the NaCl-induced Na⁺/H⁺ exchange in root tissues and cells was inhibited by amiloride (a Na⁺/H⁺ antiporter inhibitor) or sodium orthovanadate (a plasma membrane H⁺-ATPase inhibitor). These results indicate that the Na⁺ extrusion in stressed P. euphratica roots is the result of an active Na⁺/H⁺ antiport across the plasma membrane. In comparison, the Na⁺/H⁺ antiport system in salt-stressed P. popularis roots was insufficient to exclude Na⁺ at both the tissue and cellular levels. Moreover, salt-treated P. euphratica roots retained a higher capacity for Cl⁻ exclusion than P. popularis, especially during a long term in high salinity. The pattern of NaCl-induced fluxes of H⁺, Na⁺, and Cl⁻ differs from that caused by isomotic mannitol in P. euphratica roots, suggesting that NaCl-induced alternations of root ion fluxes are mainly the result of ion-specific effects.Soil salinity causes increasingly agricultural and environmental problems on a worldwide scale, especially in arid areas. When plant roots are subjected to saline environments with high NaCl content, external Na⁺ and Cl⁻ establish a large electrochemical gradient favoring the passive entry of salt ions through a variety of cation and anion channels and/or transporters in the plasma membrane (PM; Blumwald et al., 2000; Hasegawa et al., 2000; White and Broadley, 2001; Roberts, 2006; Demidchik and Maathuis, 2007). The entry and accumulation of toxic ions lead to disruption of ion homeostasis and finally cause secondary stress, e.g. oxidative bursts (Zhu, 2001, 2003). Accordingly, the maintenance of low salt concentration in the cytosol is of great importance for salt adaptation of plants (Greenway and Munns, 1980; Munns and Tester, 2008).Active Na⁺ extrusion to the apoplast or external environment is essential for sustaining Na⁺ homeostasis in the cytosol (Blumwald et al., 2000; Tester and Davenport, 2003; Zhu, 2003; Apse and Blumwald, 2007). PM Na⁺/H⁺ antiporters have been widely considered to play a crucial role in active Na⁺ extrusion under saline conditions (Shi et al., 2000, 2002; Qiu et al., 2002; Martínez-Atienza et al., 2007). NaCl-induced activity of PM Na⁺/H⁺ antiporter has been reported in crop species, tomato (Solanum lycopersicum; Wilson and Shannon, 1995), Arabidopsis (Arabidopsis thaliana; Qiu et al., 2002, 2003), and rice (Oryza sativa; Martínez-Atienza et al., 2007). Furthermore, overexpression of the Na⁺/H⁺ antiporter gene AtSOS1 decreases the accumulation of Na⁺ in transgenic Arabidopsis under NaCl stress (Shi et al., 2003). These PM Na⁺/H⁺ antiporters depend on electrochemical H⁺ gradients, which are generated by PM H⁺-ATPase (Blumwald et al., 2000; Zhu, 2003). Using an ion-selective microelectrode, Shabala and a colleague suggested the involvement of PM H⁺-ATPase in the Na⁺/H⁺ antiport according to H⁺ kinetics on salt shock (Shabala, 2000; Shabala and Newman, 2000). Therefore, the NaCl-induced H⁺ pumping is fundamental to Na⁺/H⁺ exchange and salinity tolerance (Ayala et al., 1996; Vitart et al., 2001; Chen et al., 2007; Gévaudant et al., 2007). However, the active Na⁺/H⁺ antiport across PM and the contribution to salt exclusion have been rarely investigated in tree species.Munns and Tester (2008) claimed that Cl⁻ toxicity is more important than Na⁺ toxicity in some woody species, e.g. citrus. Similarly, we have noticed that the inability to restrict Cl⁻ uptake contributes to the NaCl-induced salt damage in salt-sensitive poplar (Populus spp.) species, in addition to toxicity of excess Na⁺ (Chen et al., 2001, 2002, 2003). The differences in Cl⁻ tolerance exhibited by plants are usually related to the ability to restrict Cl⁻ transport to the aerial part (Greenway and Munns, 1980; White and Broadley, 2001). Excluding Cl⁻ from the xylem seems to be an effective mechanism for lotus to cope with the interactive effect of salt and water logging (Teakle et al., 2007). An influx of Cl⁻, immediately after addition of NaCl, was observed in bean (Vicia faba) mesophyll tissue (Shabala, 2000). The Cl⁻ flux response to salt shock is helpful to reveal the rapid adjustments of plants to salinity. However, Cl⁻ fluxes in salt-adapted roots, which are necessary to clarify plant adaptations to long durations of salinity, have not been examined.Populus euphratica has been widely considered as a model plant to elucidate physiological and molecular mechanisms of salt tolerance in woody species (Chen et al., 2001, 2002, 2003; Gu et al., 2004; Ottow et al., 2005a, 2005b; Junghans et al., 2006; Wang et al., 2007, 2008; Wu et al., 2007; Zhang et al., 2007). Comparative studies have shown that salt-stressed P. euphratica seedlings accumulate less Na⁺ and Cl⁻ in root and shoot tissues than salt-sensitive poplar species (Chen et al., 2001, 2002). It is suggested that the greater capacity to exclude NaCl in P. euphratica is likely the result of salt uptake and transport restriction in roots (Chen et al., 2002, 2003). However, this needs further investigations, e.g. by electrophysiology, to clarify.In this study, we used a noninvasive ion flux technique to measure the tissue and cellular fluxes of H⁺, Na⁺, and Cl⁻ in roots of the salt-tolerant P. euphratica and salt-sensitive P. popularis 35-44. The aim was to compare the NaCl-induced alternations of ion fluxes in poplar species differing in salt tolerance. Furthermore, we examined the effects of pH, salt shock, and PM transport inhibitors on Na⁺ and H⁺ fluxes in root-derived protoplasts of the salt-tolerant species, P. euphratica, which exhibited an evident Na⁺ exclusion under saline conditions.     

Cerium oxide nanoparticles protect against Aβ-induced mitochondrial fragmentation and neuronal cell death

Evidence indicates that nitrosative stress and mitochondrial dysfunction participate in the pathogenesis of Alzheimer''s disease (AD). Amyloid beta (Aβ) and peroxynitrite induce mitochondrial fragmentation and neuronal cell death by abnormal activation of dynamin-related protein 1 (DRP1), a large GTPase that regulates mitochondrial fission. The exact mechanisms of mitochondrial fragmentation and DRP1 overactivation in AD remain unknown; however, DRP1 serine 616 (S616) phosphorylation is likely involved. Although it is clear that nitrosative stress caused by peroxynitrite has a role in AD, effective antioxidant therapies are lacking. Cerium oxide nanoparticles, or nanoceria, switch between their Ce³⁺ and Ce⁴⁺ states and are able to scavenge superoxide anions, hydrogen peroxide and peroxynitrite. Therefore, nanoceria might protect against neurodegeneration. Here we report that nanoceria are internalized by neurons and accumulate at the mitochondrial outer membrane and plasma membrane. Furthermore, nanoceria reduce levels of reactive nitrogen species and protein tyrosine nitration in neurons exposed to peroxynitrite. Importantly, nanoceria reduce endogenous peroxynitrite and Aβ-induced mitochondrial fragmentation, DRP1 S616 hyperphosphorylation and neuronal cell death.Nitric oxide (NO) is a neurotransmitter and neuromodulator required for learning and memory.¹ NO is generated by NO synthases, a group of enzymes that produce NO from L-arginine. In addition to its normal role in physiology, NO is implicated in pathophysiology. When overproduced, NO combines with superoxide anions (O₂·⁻), byproducts of aerobic metabolism and mitochondrial oxidative phosphorylation, to form peroxynitrite anions (ONOO⁻) that are highly reactive and neurotoxic. Accumulation of these reactive oxygen species (ROS) and reactive nitrogen species (RNS), known as oxidative and nitrosative stress, respectively, is a common feature of aging, neurodegeneration and Alzheimer''s disease (AD).¹Nitrosative stress caused by peroxynitrite has a critical role in the etiology and pathogenesis of AD.^{2, 3, 4, 5, 6, 7} Peroxynitrite is implicated in the formation of the two hallmarks of AD, Aβ aggregates and neurofibrillary tangles containing hyperphosphorylated Tau protein.^{1, 4, 7} In addition, peroxynitrite promotes the nitrotyrosination of presenilin 1, the catalytic subunit of the γ-secretase complex, which shifts production of Aβ to amyloid beta (Aβ)42 and increases the Aβ42/Aβ40 ratio, ultimately resulting in an increased propensity for aggregation and neurotoxicity.⁵ Furthermore, nitration of Aβ tyrosine 10 enhances its aggregation.⁶ Peroxynitrite can also modify enzymes, such as triosephosphate isomerase,⁴ and activate kinases, including Jun amino-terminal kinase and p38 mitogen-activated protein kinase, which enhance neuronal cell death.^{8, 9} Moreover, peroxynitrite can trigger the release of free metals such as Zn²⁺ from intracellular stores with consequent inhibition of mitochondrial function and enhancement of neuronal cell death.^{10, 11, 12} Finally, peroxynitrite can irreversibly inhibit complexes I and IV of the mitochondrial respiratory chain.^{11, 13}Because mitochondria have a critical role in neurons as energy producers to fuel vital processes such as synaptic transmission and axonal transport,¹⁴ and mitochondrial dysfunction is a well-documented and early event in AD,¹⁵ it is important to consider how peroxynitrite and nitrosative stress affect mitochondria. Although the ultimate cause of mitochondrial dysfunction in AD remains unclear, an imbalance in mitochondrial fission and fusion is one possibility.^{1, 14, 16, 17, 18} Notably, peroxynitrite, N-methyl D-aspartate (NMDA) receptor activation and Aβ can induce mitochondrial fragmentation by activating mitochondrial fission and/or inhibiting fusion.¹⁶ Mitochondrial fission and fusion is regulated by large GTPases of the dynamin family, including dynamin-related protein 1 (DRP1) that is required for mitochondrial division,¹⁹ and inhibition of mitochondrial division by overexpression of the GTPase-defective DRP1^K38A mutant provides protection against peroxynitrite-, NMDA- and Aβ-induced mitochondrial fragmentation and neuronal cell death.¹⁶The exact mechanism of peroxynitrite-induced mitochondrial fragmentation remains unclear. A recent report suggested that S-nitrosylation of DRP1 at cysteine 644 increases DRP1 activity and is the cause of peroxynitrite-induced mitochondrial fragmentation in AD;²⁰ however, the work remains controversial, suggesting that alternative pathways might be involved.²¹ For example, peroxynitrite also causes rapid DRP1 S616 phosphorylation that promotes its translocation to mitochondria and organelle division.^{21, 22} In mitotic cells, DRP1 S616 phosphorylation is mediated by Cdk1/cyclinB1 and synchronizes mitochondrial division with cell division.²³ Interestingly, DRP1 is S616 hyperphosphorylated in AD brains, suggesting that this event might contribute to mitochondrial fragmentation in the disease.^{21, 22} A recent report indicates that Cdk5/p35 is responsible for DRP1 S616 phosphorylation,²⁴ and notably aberrant Cdk5/p35/p25 signaling is associated with AD pathogenesis.²⁵ Thus, we explored here the possible role of DRP1 S616 hyperphosphorylation in Aβ- and peroxynitrite-mediated mitochondrial fragmentation.Under normal conditions, accumulated mitochondrial superoxide anions and hydrogen peroxide (H₂O₂) can be neutralized by superoxide dismutase (SOD) and catalase. Nitrosative stress in aging and AD might be explained by a loss of antioxidant enzymes. Previous studies suggest that expression of SOD subtypes is decreased in the human AD brain.^{26, 27} Furthermore, SOD1 deletion in a mouse model of AD increased the burden of amyloid plaques.²⁶ By contrast, overexpression of SOD2 in a mouse model of AD decreased the Aβ42/Aβ40 ratio and alleviated memory deficits.^{28, 29} There is currently a lack of antioxidants that can effectively quench superoxide anions, H₂O₂ or peroxynitrite and provide lasting effects. Cerium is a rare earth element and cerium oxide (CeO₂) nanoparticles, or nanoceria, shuttle between their 3+ or 4+ states. Oxidation of Ce⁴⁺ to Ce³⁺ causes oxygen vacancies and defects on the surface of the crystalline lattice structure of the nanoparticles, generating a cage for redox reactions to occur.³⁰ Accordingly, nanoceria mimic the catalytic activities of antioxidant enzymes, such as SOD^{31, 32} and catalase,³³ and are able to neutralize peroxynitrite.³⁴ Because of these antioxidant properties, we hypothesized that nanoceria could detoxify peroxynitrite and protect against Aβ-induced DRP1 S616 hyperphosphorylation, mitochondrial fragmentation and neuronal cell death.     

Transforming growth factor-beta signaling network regulates plasticity and lineage commitment of lung cancer cells

Identification of target cells in lung tumorigenesis and characterization of the signals that control their behavior is an important step toward improving early cancer diagnosis and predicting tumor behavior. We identified a population of cells in the adult lung that bear the EpCAM+CD104+CD49f+CD44+CD24loSCA1+ phenotype and can be clonally expanded in culture, consistent with the properties of early progenitor cells. We show that these cells, rather than being restricted to one tumor type, can give rise to several different types of cancer, including adenocarcinoma and squamous cell carcinoma. We further demonstrate that these cells can be converted from one cancer type to the other, and this plasticity is determined by their responsiveness to transforming growth factor (TGF)-beta signaling. Our data establish a mechanistic link between TGF-beta signaling and SOX2 expression, and identify the TGF-beta/SMAD/SOX2 signaling network as a key regulator of lineage commitment and differentiation of lung cancer cells.Lung cancer is the leading cause of cancer-related mortality in both men and women worldwide. Lung cancers are divided into two major categories: non-small-cell lung cancer (NSCLC) and small-cell lung cancer. NSCLC accounts for ∼80% of all lung cancers and is divided further into adenocarcinoma (ADC), squamous cell carcinoma (SCC) and large-cell lung carcinoma. Of the four major types of lung cancer, Kras mutations are present in about 30–50% of ADC, a smaller percentage of SCC (5–7%) and <1% of SCLC.^{1, 2} Mutations of the p53 gene are common in all types of lung cancer and range from ∼30% in ADC to more than 70% in SCC and SCLC.³ Other alterations occur at lower frequencies in NSCLC, including mutations in EGFR (15%), EML4-ALK (4%), ERBB2 (2%), AKT1, BRAF, MAP2K1 and MET.^{2, 4} Previous efforts in comprehensive characterization of lung cancer include copy number and gene expression profiling, targeted sequencing of candidate genes and large-scale genome sequencing of tumor samples.^{5, 6, 7, 8, 9} Significant progress has also been made in developing mouse models of lung carcinogenesis.^{10, 11} The unifying theme underlying these studies is that there exists a permissive cellular context for each specific oncogenic lesion, and that only certain types of cells are capable of cancer initiation.^{12, 13, 14}The lung consists of three anatomically distinct regions such as trachea, bronchioles and alveoli, each maintained by a distinct population of progenitor cells, that is, basal, Clara and alveolar type 2 (AT2) cells, respectively.^{15, 16} Previous work has focused upon AT2 cells, Clara cells (or variant Clara cells with low CC10 expression) and the putative bronchioalveolar stem cells (BASCs) as potential cells of origin for lung ADC.^{12, 14, 17} However, to date, only AT2 cells have been conclusively identified as having the potential to be the cells of origin for lung ADC.^{14, 17} This raises the question of whether Clara cells, their restricted subpopulations or the newly identified candidate stem cells, termed distal airway stem cells,¹⁸ alveolar epithelial progenitor cells (AECs)^{19, 20} and BASCs,¹² also have the capacity to give rise to ADC. Current knowledge on the cellular origins of SCC, the second most common type of lung cancer, lags behind that of ADC, partly owing to the fact that squamous cells are not normally present in the respiratory epithelium, and therefore arise through either metaplasia (conversions between stem cell states) or trans-differentiation (conversions between differentiated cells).^{21, 22} Whether the mechanisms of SCC causation vary by cell type, their responses to various cells signaling cascades (e.g., transforming growth factor (TGF)-beta, WNT, etc.), or other tumor characteristics is unknown at present.To address the questions of cell type of origin and signal cascades that control their behavior, we developed in vitro culture conditions that favor the growth of lung epithelial cells with stem cell-like properties. We describe a population of cells isolated from the adult lung that, rather than being restricted to one tumor type, can give rise to several different types of cancer, including ADC and SCC. We also show that these cells can be converted from one cancer type to the other, and this plasticity is largely, if not solely, determined by TGF-beta signaling.     

Decreased mitochondrial priming determines chemoresistance of colon cancer stem cells

Tumor heterogeneity is in part determined by the existence of cancer stem cells (CSCs) and more differentiated tumor cells. CSCs are considered to be the tumorigenic root of cancers and suggested to be chemotherapy resistant. Here we exploited an assay that allowed us to measure chemotherapy-induced cell death in CSCs and differentiated tumor cells simultaneously. This confirmed that CSCs are selectively resistant to conventional chemotherapy, which we revealed is determined by decreased mitochondrial priming. In agreement, lowering the anti-apoptotic threshold using ABT-737 and WEHI-539 was sufficient to enhance chemotherapy efficacy, whereas ABT-199 failed to sensitize CSCs. Our data therefore point to a crucial role of BCLXL in protecting CSCs from chemotherapy and suggest that BH3 mimetics, in combination with chemotherapy, can be an efficient way to target chemotherapy-resistant CSCs.Colorectal cancer is the third most common cancer worldwide.^{1, 2} Patients with advanced stage colorectal cancer are routinely treated with 5-fluorouracil (5-FU), leucovorin and oxaliplatin (FOLFOX), or with 5-FU, leucovorin and irinotecan (FOLFIRI), often in combination with targeted agents such as anti-VEGF or anti-EGFR at metastatic disease.^{3, 4, 5, 6} Despite this intensive treatment, therapy is still insufficiently effective and chemotherapy resistance occurs frequently. Although still speculative, it has been suggested that unequal sensitivity to chemotherapy is due to an intratumoral heterogeneity that is orchestrated by cancer stem cells (CSCs) that can self-renew and give rise to more differentiated progeny.^{7, 8} When isolated from patients, CSCs efficiently form tumors upon xenotransplantation into mice which resemble the primary tumor from which they originated.^{9, 10, 11} In addition, many xenotransplantation studies have emphasized the importance of CSCs for tumor growth.^{9, 10, 11, 12} Colon CSCs were originally isolated from primary human colorectal tumor specimens using CD133 as cell surface marker and shown to be highly tumorigenic when compared with the non-CSCs population within a tumor.^{9, 10} Later, other cell surface markers as well as the activity of the Wnt pathway have been used to isolate colon CSCs from tumors.^{12, 13} Spheroid cultures have been established from human primary colorectal tumors that selectively enrich for the growth of colon CSCs,^{11, 12} although it is important to realize that these spheres also contain more differentiated tumor cells.¹² In agreement, we have shown that the Wnt activity reporter that directs the expression of enhanced green fluorescent protein (TOP-GFP) allows for the separation of CSCs from more differentiated progeny in the spheroid cultures.¹²CSCs are suggested to be responsible for tumor recurrence after initial therapy, as they are considered to be selectively resistant to therapy.^{11, 14} Conventional chemotherapy induces, among others, DNA damage and subsequent activation of the mitochondrial cell death pathway, which is regulated by a balance between pro- and anti-apoptotic BCL2 family members.¹⁵ Upon activation of apoptosis, pro-apoptotic BH3 molecules are activated and these may perturb the balance in favor of apoptosis initiated by mitochondrial outer membrane polarization (MOMP), release of cytochrome c and subsequent activation of a caspase cascade.The apoptotic balance of cancer cells can be measured with the use of a functional assay called BH3 profiling.¹⁶ BH3 profiling is a method to determine the apoptotic ‘priming'' level of a cell by exposing mitochondria to standardized amounts of roughly 20-mer peptides derived from the alpha-helical BH3 domains of BH3-only proteins and determining the rate of mitochondrial depolarization. Using this approach, priming was measured in various cancers and compared with normal tissues.^{17, 18} In all cancer types tested, the mitochondrial priming correlated well with the observed clinical response to chemotherapy. That is, cancers that are highly primed are more chemosensitive, whereas chemoresistant tumor cells and normal tissues are poorly primed.^{17, 18} This suggests that increasing mitochondrial priming can potentially increase chemosensitivity, which can be achieved by directly inhibiting the anti-apoptotic BCL2 family members.¹⁸ To this end, small-molecule inhibitors, so-called BH3 mimetics, have been developed. ABT-737 and the highly related ABT-263 both inhibit BCL2, BCLXL and BCLW^{19, 20, 21} and were shown to be effective in killing cancer cells in vitro and in vivo²¹ with a preference for BCL2.^{19, 22} As BCL2 protein expression is often upregulated in hematopoietic cancers, it represents a promising target, which was supported by high efficacy of these BH3 mimetics in animal experiments.²¹ However, in vivo efficacy is limited due to thrombocytopenia, which relates to a dependence of platelets on BCLXL for survival.^{23, 24} To overcome this toxicity, a BCL2-specific compound, ABT-199, was developed.²⁵ Souers et al.²⁵ showed that inhibition of BCL2 using ABT-199 blocks tumor growth of acute lymphoblastic leukemia cells in xenografts. In addition to the single compound effects of ABT-199, combination with rituximab inhibited growth of non-Hodgkin''s lymphoma, mantle cell lymphoma and acute lymphoblastic leukemia tumor cells growth in vivo.²⁵ Moreover, highly effective tumor lysis was observed in all three patients with chronic lymphocytic leukemia that were treated with ABT-199.²⁵ More recently, a BCLXL-specific compound, WEHI-539, was discovered using high-throughput chemical screening.²⁶As the apoptotic balance appears a useful target for the treatment of cancers and CSCs have been suggested to resist therapy selectively, we set out to analyze whether specifically colon CSCs are resistant to therapy and whether this is due to an enhanced anti-apoptotic threshold, specific to CSCs. To study chemosensitivity, we developed a robust single cell-based analysis in which we can measure apoptosis simultaneously in CSCs and their differentiated progeny. Utilizing this system we showed that colon CSCs and not their differentiated progeny are resistant to chemotherapeutic compounds and that this was due to the fact that these cells are less primed to mitochondrial death. Furthermore, inhibition of anti-apoptotic BCLXL molecule with either ABT-737 or WEHI-539, but not ABT-199, breaks this resistance and sensitizes the CSCs to chemotherapy.