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.     

NAMPT inhibition sensitizes pancreatic adenocarcinoma cells to tumor-selective,PAR-independent metabolic catastrophe and cell death induced by β-lapachone

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD⁺ synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD⁺ pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD⁺ consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD⁺ synthesis while increasing NAD⁺ consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)⁺ depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD⁺-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD⁺ synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD⁺ pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.An emerging metabolic target for the treatment of recalcitrant cancers, such as pancreatic adenocarcinoma (PDA), is their reliance on NAD⁺ synthesis, particularly through the nicotinamide-recycling pathway.^{1, 2, 3} Rapid and efficient NAD⁺ synthesis is critical to sustain signaling processes, such as deacetylation by sirtuins and adenosine diphosphate (ADP) ribosylation by poly(ADP ribose) polymerase 1 (PARP1). NAD(P)⁺ pools are also necessary to support anabolic metabolism and proliferation of cancer cells. In an attempt to leverage increased tumor-cell reliance on NAD⁺ synthesis, small molecule inhibitors of nicotinamide phosphoribosyltransferase (NAMPT) were developed (e.g., FK866).⁴ NAMPT catalyzes the rate-limiting step of the most active pathway of NAD⁺ synthesis. Inhibitors of NAMPT, such as FK866, reduce NAD⁺ levels, induce canonical apoptosis preferentially in cancer cells in vitro, inhibit tumor growth, and increase overall survival in preclinical cancer models.^{1, 5, 6, 7} FK866 (APO866) was relatively well tolerated in humans and advanced to phase II clinical trials. However, owing to its short half-life in circulation, prolonged treatment regimens were required and toxicity to normal, rapidly proliferating hematopoietic cells was noted. Accordingly, FK866 and other NAMPT inhibitors did not demonstrate sufficient tumor-selectivity to achieve clinical success as single agents.⁸To increase the specificity and efficacy of NAMPT inhibition, we combined FK866 with β-lapachone (β-lap), a targeted cancer therapeutic that causes tumor-selective PARP1 hyperactivation and NAD⁺ depletion in an NADPH:quinone oxidoreductase 1 (NQO1)-specific manner.⁹ β-Lap is a substrate for two-electron oxidoreduction by NQO1, a Phase II quinone-detoxifying enzyme.⁹ The resulting hydroquinone form of β-lap is highly unstable and spontaneously reacts with oxygen to revert back to the parent compound, generating two moles of superoxide per mole of NAD(P)H used in the process. This results in a futile cycle that occurs rapidly in NQO1-overexpressing cells, causing marked NADH/NADPH oxidation. DNA damage in the form of base oxidation and DNA single-strand breaks results from H₂O₂ generated from the futile redox cycle. In an attempt to repair this damage, PARP1 becomes hyperactivated, generating extensive branched poly(ADP ribose) (PAR) polymer. Hyperactivated PARP1 substantially depletes NAD⁺ and ultimately adenosine triphosphate (ATP) levels, thereby inhibiting subsequent repair of β-lap-induced DNA lesions. The observed cell death is caspase-independent and driven by nuclear translocation of apoptosis-inducing factor (AIF), activation of μ-calpain, and post-translational modification of GAPDH.^{10, 11, 12, 13} NQO1 is highly expressed in many types of cancer, and the therapeutic window provided by NQO1 bioactivation of β-lap has advanced its use to phase I clinical trials (ARQ761).¹⁴ Elevated NQO1 expression (≥10-fold) has been identified in ~85% of patient tissue from pancreatic ductal adenocarcinoma (PDA), making pancreatic cancer an especially appealing target for therapy using NQO1 bioactivatable drugs, such as β-lap.^{15, 16, 17, 18} However, dose-limiting methemoglobinemia caused by nonspecific reactive oxygen species (ROS) generation at high β-lap doses may limit the efficacy of β-lap as monotherapy.¹⁹ Strategies for increasing cancer cell cytotoxicity while maintaining NQO1 specificity could enhance use of β-lap for therapy against PDAs, as well as other solid cancers that overexpress NQO1.We found that examining cell death pathways induced by β-lap, with or without FK866 treatment, is a novel means to elucidate general mechanisms of lethality mediated by NAD⁺ loss, as cell death by PARP1 hyperactivation occurs in other contexts. Notably, cell death induced by ischemia/reperfusion shares many of the same characteristics: ROS induction, PARP1 hyperactivation, calcium release, AIF translocation, and caspase-independence.^{20, 21} Similarly, treatment with methylnitronitrosoguanidine (MNNG; a DNA alkylating agent) or induction of neuronal excitotoxicty induces PARP1 hyperactivation and cell death, but without futile cycle-induced ROS production.^{22, 23, 24} Recent studies suggest an important role for accumulated free PAR polymer that can directly activate μ-calpain, activate and release AIF, and inhibit glycolysis.^{22, 25, 26, 27, 28} By combining β-lap and FK866, we uncouple NAD⁺ and ATP depletion from the robust formation of PAR noted with β-lap alone, allowing us to define the function of PAR formation in β-lap-induced cell death.β-Lap and FK866 have distinct, but highly complementary mechanisms of action. β-Lap induces tumor-selective NAD⁺ depletion specifically in cancer cells that express high levels of NQO1. FK866 primes cancer cells for cell death by lowering NAD⁺/NADH pools and prevents recovery by inhibiting NAD⁺ synthesis from nicotinamide liberated by activated PARP1. We show that the increased dependence of PDA cells on glycolysis is specifically targeted by ROS-induced, NAD⁺ depletion caused by exposure to both drugs. Glycolytic inhibition, ATP depletion, and cell death is independent of PAR formation, strongly suggesting that PAR accumulation is not directly involved. The use of β-lap with NAMPT inhibitors results in synergistic NQO1- and PARP1-dependent cancer cell death, allowing the use of lower doses and shorter treatment times for both therapeutics.     

Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner

Cellular necrosis has long been regarded as an incidental and uncontrolled form of cell death. However, a regulated form of cell death termed necroptosis has been identified recently. Necroptosis can be induced by extracellular cytokines, pathogens and several pharmacological compounds, which share the property of triggering the formation of a RIPK3-containing molecular complex supporting cell death. Of interest, most ligands known to induce necroptosis (including notably TNF and FASL) can also promote apoptosis, and the mechanisms regulating the decision of cells to commit to one form of cell death or the other are still poorly defined. We demonstrate herein that intracellular nicotinamide adenine dinucleotide (NAD⁺) has an important role in supporting cell progression to necroptosis. Using a panel of pharmacological and genetic approaches, we show that intracellular NAD⁺ promotes necroptosis of the L929 cell line in response to TNF. Use of a pan-sirtuin inhibitor and shRNA-mediated protein knockdown led us to uncover a role for the NAD⁺-dependent family of sirtuins, and in particular for SIRT2 and SIRT5, in the regulation of the necroptotic cell death program. Thus, and in contrast to a generally held view, intracellular NAD⁺ does not represent a universal pro-survival factor, but rather acts as a key metabolite regulating the choice of cell demise in response to both intrinsic and extrinsic factors.Nicotinamide adenine dinucleotide (NAD⁺) has been long recognized as a key intermediate in cellular metabolism. By accepting and donating electrons in reactions catalyzed by dehydrogenases, NAD⁺ has, for example, a central role in the generation of ATP, a molecule required for most energy-consuming cellular reactions. The recognition of NAD⁺ as a substrate in a number of regulatory processes has shed a new light on its role in cell physiology. Indeed, NAD⁺ represents a substrate for a wide range of enzymes including cADP-ribose synthases, poly (ADP-ribose) polymerases (PARPs) and the sirtuin family of NAD⁺-dependent deacylases (SIRTs). In marked contrast to its role in energy metabolism, the involvement of NAD⁺ in these enzymatic reactions is based on its ability to function as a donor of ADP-ribose, a reaction that, if sustained, can lead to the depletion of the intracellular NAD⁺ pool.^{1, 2, 3, 4, 5}The pro-survival role of NAD⁺ has been particularly well described in cells exposed to genotoxic/oxidative stress. In response to DNA damage, PARP1, the founding and most abundant member of the PARP family, binds to DNA strand breaks and initiates a repair response by catalyzing the post-translational modification of several nuclear proteins, including itself. This protective response is characterized by the transfer of successive units of the ADP-ribose moiety (up to 200 units) from NAD⁺ to other proteins, compromising therefore both energy production (slowing the rate of glycolysis, electron transport and ATP formation) and activity of other NAD⁺-dependent enzymes through NAD⁺ depletion.^{6, 7} Moreover, PARP1-synthesized PAR polymers can be degraded into free oligomers, known to translocate to the mitochondria where they can trigger the release of AIF from mitochondria to the nucleus.^{8, 9, 10, 11} The precise molecular steps linking PARP1 activation to this form of stress-induced cell death, termed parthanatos, have not been fully elucidated, and probably depend on the particular metabolic status of the cell examined (i.e., anerobic glycolysis in most in vitro cell lines versus oxidative metabolism of neuronal cells, see Welsby et al.¹² for review). In any instances, and independently of the fine molecular events at work, virtually all studies have identified the brisk loss of intracellular NAD⁺ as the critical step initiating this specific form of cell death. The protective role of NAD⁺ in PARP1-dependent cell death has been indeed amply documented.^{13, 14, 15, 16, 17, 18} In mammals, nicotinamide (Nam) acts as the main precursor for NAD⁺ biosynthesis and nicotinamide phosphoribosyl tranferase (NAMPT) is the rate-limiting enzyme for NAD⁺ biosynthesis from Nam.¹⁹ Nampt deficiency in mice leads to lethality and the heterozygous animals suffer from significant perturbations related to their NAD⁺ metabolism.²⁰ In keeping with the general role of NAD⁺ as a survival factor in cells exposed to genotoxic stress, genetic ablation of Nampt and/or treatment with a specific NAMPT inhibitor (FK866) sensitized cells to the toxic effects of alkylating agents.^{16, 18} Similarly, overexpression of a catalytically active recombinant NAMPT protected the NIH-3T3 cell line from the toxicity of the same DNA alkylating agents,¹⁸ further establishing a functional link between NAD⁺ biosynthesis and sensitivity to stress-induced, PARP1-dependent cell death.While analyzing the influence of NAD metabolism on survival of NIH3T3 cells exposed to genotoxic agents, we observed that overexpression of NAMPT prolonged cell survival of cells exposed to the alkylating agent N-methyl-N''-nitro-N-nitrosoguanidine (MNNG), and unexpectedly, led to increased sensitivity to cell death induced by the pro-inflammatory cytokine TNF.¹⁸ TNF is a pleiotropic cytokine regulating many cellular functions and known to induce several forms of cell death, including apoptosis and the recently uncovered regulated form of necrosis termed necroptosis.^{21, 22} In contrast to apoptosis, necroptosis is largely independent of the so-called executioner caspase (such as caspase-3, 6 and 7) activity and is initiated by the formation of a signaling complex comprising the receptor-interacting serine-threonine kinase 1 (RIPK1), RIPK3 and the recently identified mixed lineage kinase domain-like protein MLKL. Although necroptosis often appears to occur when apoptosis is abortive (such as in situations of caspase inhibition), the cellular factors regulating the choice between these two forms of regulated cell death have not been fully uncovered. Using a model cell line engineered to respond to both apoptosis and necroptosis, we demonstrate herein that intracellular NAD⁺ represents a critical factor in promoting cell death by necroptosis. In keeping with the well-described role of sirtuins as intracellular NAD⁺ sensors, we also demonstrate that sirtuins, and in particular SIRT2 and SIRT5, are required for adequate completion of the necroptotic program in response to TNF. Accordingly, a pan-sirtuin inhibitor was found to attenuate organ damage induced by transient ischemia. Thus, intracellular NAD⁺, rather than acting as a general cell survival factor, appears to promote cell necroptosis in a sirtuin-dependent manner, a finding that may suggest novel therapeutic approaches to attenuate in vivo necrotic insults in several pathological settings.     

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.     

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.     

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.     

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.     

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.     

Heme oxygenase-1 protects against Alzheimer's amyloid-β1-42-induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer''s disease, catabolises heme to biliverdin, Fe²⁺ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediates cellular K⁺ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ_1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrP^c, and rat primary hippocampal neurons. Aβ_1-42 (containing protofibrils) caused a concentration-dependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrP^c-expressing cells showing greater susceptibility to Aβ_1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ_1-42. The CO-donor CORM-2 protected cells against Aβ_1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ_1-42 treatment suggesting that K⁺ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca²⁺ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent.Amongst the earliest of events leading to neuronal loss in Alzheimer''s disease (AD) is the loss of functional synapses,^{1, 2, 3} apparent long before deposition of amyloid β peptide (Aβ)-containing plaques.⁴ Although other parts of the neurone (e.g. the axon or soma) appear intact, their health at this early stage of disease progression is not clear. However, neurones ultimately die in AD and there is clear evidence that numerous events indicative of apoptosis occur even at early stages of disease progression.^{5, 6, 7, 8} Thus, targeting of apoptotic mechanisms may be of therapeutic value in AD as well as in other neurodegenerative disorders. Furthermore, apoptosis is established as a mechanism of neuronal loss following other types of pathological stresses including ischemia associated with stroke,⁹ which can predispose individuals to the development of AD.^{10, 11, 12}Apoptosis is strongly influenced by intracellular K⁺ levels¹³ which regulate caspase activation, mitochondrial membrane potential and volume, osmolarity and cell volume.^{13, 14} K⁺ loss via K⁺ channels is a key early stage in apoptosis,^{15, 16, 17, 18, 19} and K⁺ channel inhibitors can protect against apoptosis triggered by numerous insults including oxidative stress.^{20, 21} Evidence suggests a particularly important role for the voltage-gated channel Kv2.1 in this process: expression of dominant negative Kv2.1 constructs (thus lacking functional Kv2.1 channels) protects against oxidant-induced apoptosis, and over-expression of Kv2.1 increases susceptibility to apoptosis.^{22, 23} Pro-apoptotic agents cause a rapid increase in the surface expression of Kv2.1 channels,²⁴ but whether or not this occurs in AD remains to be determined. Alternative pathways recently reported to promote cell death include activation of the AMP-dependent protein kinase (AMP kinase) which can act either as a Tau kinase²⁵ or to inhibit the mTOR pathway²⁶ and thus contribute to neurodegeneration.Heme oxygenases (HO) are enzymes widely distributed throughout the body. In the central nervous system, HO-2 is constitutively expressed in neurones and astrocytes, while HO-1 is inducible in both cell types.^{27, 28, 29, 30} Both HO-1 and HO-2 break down heme to liberate biliverdin, ferrous iron (Fe²⁺) and carbon monoxide (CO). This catalysis is of biological significance since it is crucial to iron and bile metabolism, and also generates a highly effective antioxidant in bilirubin (from biliverdin via bilirubin reductase). Numerous stimuli can induce HO-1 gene expression,³¹ including oxidative stress³² and Aβ peptides.³³ Importantly, HO-1 is strikingly up-regulated in AD patients, a finding considered indicative of oxidative stress.^{27, 34, 35} Induction of HO-1 is clearly a neuroprotective response (although in some cases can exert detrimental effects²⁷). However, there is growing evidence that CO can be neuroprotective, for example against the damage of focal ischemia.³⁶ Our recent studies have demonstrated that CO provides protection against oxidant-induced apoptosis by selectively inhibiting Kv2.1.^{23, 37} In the present study, we have investigated whether HO-1, or its product CO, can provide protection against Aβ-induced toxicity in the human neuroblastoma, SH-SY5Y, and in rat primary hippocampal neurones, and whether this involves regulation of K⁺ channels. We show that both HO-1 and CO protect cells against the toxicity of protofibrillar Aβ_1-42 but that protection does not arise from inhibition of apoptosis-associated K⁺ efflux, but rather by inhibition of AMPK activation.     

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.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

Mesenchymal stem cells stimulate intestinal stem cells to repair radiation-induced intestinal injury

The loss of stem cells residing in the base of the intestinal crypt has a key role in radiation-induced intestinal injury. In particular, Lgr5⁺ intestinal stem cells (ISCs) are indispensable for intestinal regeneration following exposure to radiation. Mesenchymal stem cells (MSCs) have previously been shown to improve intestinal epithelial repair in a mouse model of radiation injury, and, therefore, it was hypothesized that this protective effect is related to Lgr5⁺ ISCs. In this study, it was found that, following exposure to radiation, transplantation of MSCs improved the survival of the mice, ameliorated intestinal injury and increased the number of regenerating crypts. Furthermore, there was a significant increase in Lgr5⁺ ISCs and their daughter cells, including Ki67⁺ transient amplifying cells, Vil1⁺ enterocytes and lysozyme⁺ Paneth cells, in response to treatment with MSCs. Crypts isolated from mice treated with MSCs formed a higher number of and larger enteroids than those from the PBS group. MSC transplantation also reduced the number of apoptotic cells within the small intestine at 6 h post-radiation. Interestingly, Wnt3a and active β-catenin protein levels were increased in the small intestines of MSC-treated mice. In addition, intravenous delivery of recombinant mouse Wnt3a after radiation reduced damage in the small intestine and was radioprotective, although not to the same degree as MSC treatment. Our results show that MSCs support the growth of endogenous Lgr5⁺ ISCs, thus promoting repair of the small intestine following exposure to radiation. The molecular mechanism of action mediating this was found to be related to increased activation of the Wnt/β-catenin signaling pathway.The epithelium of the small intestine contains crypts and villi. Intestinal stem cells (ISCs) reside in the base of the crypts and are responsible for maintaining intestinal epithelial homeostasis and regeneration following injury.^{1, 2} Recent studies have identified two populations of stem cells in the small intestine of mice called Lgr5⁺ and Bmi1⁺ ISCs.^{3, 4, 5, 6, 7, 8, 9, 10, 11} Lgr5⁺ ISCs, also known as crypt base columnar cells (CBCs), are interspersed among the Paneth cells and are active rapidly cycling stem cells.¹² A single Lgr5⁺ ISC can grow to form ‘enteroids'' in vitro that develop into all the differentiated cell types found in the intestinal crypt.¹³ Conversely, Bmi1⁺ cells are a population of ISCs located at position +4 relative to the base of the crypt, and are quiescent, slowly cycling stem cells.¹⁴ The loss of ISCs has a critical role in radiation-induced intestinal injury (RIII).^{15, 16, 17, 18} Apoptosis of stem cells because of exposure to radiation prevents normal re-epithelialization of the intestines. Therefore, enhancing the survival of ISCs following radiation is a potential effective treatment for RIII.Mesenchymal stem cells (MSCs) possess significant potential as a therapeutic for tissue damage because of their ability to regulate inflammation, inhibit apoptosis, promote angiogenesis, and support the growth and differentiation of local stem and progenitor cells.^{19, 20} However, the mechanisms by which MSCs mediate these beneficial effects remain unclear, although it has been suggested that MSCs may actively secrete a broad range of bioactive molecules with immunomodulatory (PGE2, IDO, NO, HLA-G5, TSG-6, IL-6, IL-10 and IL-1RA), mitogenic (TGFα/β, HGF, IGF-1, bFGF and EGF), angiogenic (VEGF and TGF-β1) and/or anti-apoptotic (STC-1 and SFRP2) properties that function to modulate the regenerative environment at the site of injury.²¹ Upon re-establishment of the microenvironment following damage, the surviving endogenous stem and progenitor cells can then regenerate the injured tissue completely.Our previous study, as well as other published studies, has found that systemic administration of MSCs improves intestinal epithelial repair in an animal model of radiation injury.^{22, 23, 24, 25} Following MSC treatment, radiation-induced lesions in mice were significantly smaller than those in the control group. However, the mechanism behind this protective effect is not fully understood. Lgr5⁺ ISCs have been previously shown to be indispensable for radiation-induced intestinal regeneration.²⁶ Therefore, in this study, we tested whether the therapeutic effects of MSCs in response to RIII are related to the Lgr5⁺ population of resident ISCs.     

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.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.