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.     

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.     

Loss of Halophytism by Interference with SOS1 Expression

The contribution of SOS1 (for Salt Overly Sensitive 1), encoding a sodium/proton antiporter, to plant salinity tolerance was analyzed in wild-type and RNA interference (RNAi) lines of the halophytic Arabidopsis (Arabidopsis thaliana)-relative Thellungiella salsuginea. Under all conditions, SOS1 mRNA abundance was higher in Thellungiella than in Arabidopsis. Ectopic expression of the Thellungiella homolog ThSOS1 suppressed the salt-sensitive phenotype of a Saccharomyces cerevisiae strain lacking sodium ion (Na⁺) efflux transporters and increased salt tolerance of wild-type Arabidopsis. thsos1-RNAi lines of Thellungiella were highly salt sensitive. A representative line, thsos1-4, showed faster Na⁺ accumulation, more severe water loss in shoots under salt stress, and slower removal of Na⁺ from the root after removal of stress compared with the wild type. thsos1-4 showed drastically higher sodium-specific fluorescence visualized by CoroNa-Green, a sodium-specific fluorophore, than the wild type, inhibition of endocytosis in root tip cells, and cell death in the adjacent elongation zone. After prolonged stress, Na⁺ accumulated inside the pericycle in thsos1-4, while sodium was confined in vacuoles of epidermis and cortex cells in the wild type. RNAi-based interference of SOS1 caused cell death in the root elongation zone, accompanied by fragmentation of vacuoles, inhibition of endocytosis, and apoplastic sodium influx into the stele and hence the shoot. Reduction in SOS1 expression changed Thellungiella that normally can grow in seawater-strength sodium chloride solutions into a plant as sensitive to Na⁺ as Arabidopsis.Accompanying the production and accumulation of osmolytes and other protective molecules, an important aspect of plant responses leading to salt stress tolerance is the regulation of uptake, reexport, and control over the distribution of sodium ions (Na⁺; Hasegawa et al., 2000; Tester and Davenport, 2003). Na⁺ appear to enter the root by several pathways (Essah et al., 2003; Pardo et al., 2006), although the nature of participating genes and their interaction in pathways require further investigation. Once Na⁺ has entered the root endodermis, a tissue that represents a barrier to ions (Peng et al., 2004), it is generally assumed that the ion enters the xylem following the movement of water to aerial parts of the plant. Despite substantial efflux of Na⁺ across the plasma membrane of root cells, the net flux of Na⁺ is unidirectional from soil to roots and then to the shoot, except for possible recirculation via the phloem (Tester and Davenport, 2003). In a range of species, the severity of damaging symptoms is positively correlated with the content of Na⁺ reaching photosynthetic tissues (Davenport et al., 2005; Ren et al., 2005; Munns et al., 2006). However, halophytic species can accumulate very high amounts of Na⁺ in vacuoles, such that Na⁺ may account for most of the total cellular osmotic potential (Tester and Davenport, 2003), and the presence of Na⁺ accelerates growth in euhalophytes to some degree (Adams et al., 1998). Emerging as the major advantage of halophytes appears to be their exceptional control over Na⁺ influx combined with export mechanisms, the ability to coordinate its distribution to various tissues, and efficient sequestration of Na⁺ into vacuoles. These characteristics are of particular advantage when plants are subjected to a sudden increase of Na⁺ salts in their environment (Hasegawa et al., 2000), whereas gradual increases in Na⁺ may be tolerated even by plants that are not halophytic in nature.Na⁺-ATPases, major Na⁺ export systems in organisms such as fungi and the moss Physcomitrella patens, have not been found in higher plants (Lunde et al., 2007). In Arabidopsis (Arabidopsis thaliana), transporters of monovalent (alkali) cations, such as HKT1 (Berthomieu et al., 2003; Rus et al., 2004), members of the NHX family (Yamaguchi et al., 2005; Pardo et al., 2006), and SOS1 (for Salt Overly Sensitive 1; Shi et al., 2000, 2002, 2003), have been shown to play roles in the movement and distribution of Na⁺ ions. Studies have shown the involvement of nonselective ion channels with roles in the transport of Na⁺ ions, but the genes encoding such function(s) have not been identified (Demidchik and Maathuis, 2007). SOS1, whose deletion resulted in a strong salt-sensitivity phenotype in Arabidopsis, encodes a plasma membrane Na⁺/H⁺ antiporter involved in removing Na⁺ ions from cells (Shi et al., 2000). This efflux strategy, which may be sufficient for the survival of unicellular organisms, must be accompanied by other means of Na⁺ confinement to avoid carryover of Na⁺ between cells in futile cycles. Hence, the physiological role of a plasma membrane Na⁺/H⁺ antiporter must be embedded in the context of tissue, organ, and whole plant distribution of ions and their transporters. A recent discovery on cell layer-specific differential responses to the salt stress of root cells supported this notion (Dinneny et al., 2008).In Arabidopsis, the SOS1 gene is most strongly expressed in the epidermis of the root tip region and in cells adjacent to vascular tissues (Shi et al., 2002). Based on the salt concentration in shoot, root, and xylem sap of wild-type Arabidopsis and its sos1 knockout mutants, the SOS1 antiporter is assumed to function in Na⁺ export under severe salt stress conditions (Shi et al., 2002). However, detailed knowledge about how a Na⁺ excluder achieves salt tolerance in a multicellular eukaryote is still missing. Significantly also, even though SOS1 has been an intensely studied component of the ion homeostasis mechanism, its involvement in the exceptional salt tolerance of halophytes is not known.Thellungiella salsuginea (salt cress), which had before been called T. halophila by us, is a close relative of Arabidopsis, which has become a model to study the genetic basis of this plant''s extreme tolerance to a variety of abiotic stress factors, including salinity (Inan et al., 2004; Gong et al., 2005; Vera-Estrella et al., 2005; Volkov and Amtmann, 2006; Amtmann, 2009). Thellungiella lacks specialized morphological structures, such as salt glands or large sodium storage cells found in other halophytes, making it a useful model for studying stress tolerance mechanisms that could be applicable to further understanding or to embark on engineering of conventional crops (Inan et al., 2004). Recently, it has been reported that Thellungiella had lower net Na⁺ uptake compared with Arabidopsis. The unidirectional influx of Na⁺ ions to roots appeared to be more restricted and/or tightly controlled in Thellungiella than in Arabidopsis. To compensate for greater influx, Arabidopsis roots showed higher Na⁺ efflux (Wang et al., 2006).Here, we wished to explore the role(s) by which ThSOS1, the SOS1 homolog in Thellungiella, could be involved in shaping the halophytic character of the species using ectopic expression of the gene in yeast and in Arabidopsis and Thellungiella SOS1-RNA interference (RNAi) lines. The results identified ThSOS1 as a genetic element whose activity limits Na⁺ accumulation and affects the distribution of Na⁺ ions at high concentration, thus acting as a major tolerance determinant.     

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.     

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.     

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.     

Differential effects of P2Y1 deletion on glial activation and survival of photoreceptors and amacrine cells in the ischemic mouse retina

Gliosis of retinal Müller glial cells may have both beneficial and detrimental effects on neurons. To investigate the role of purinergic signaling in ischemia-induced reactive gliosis, transient retinal ischemia was evoked by elevation of the intraocular pressure in wild-type (Wt) mice and in mice deficient in the glia-specific nucleotide receptor P2Y₁ (P2Y₁ receptor-deficient (P2Y1R-KO)). While control retinae of P2Y1R-KO mice displayed reduced cell numbers in the ganglion cell and inner nuclear layers, ischemia induced apoptotic death of cells in all retinal layers in both, Wt and P2Y1R-KO mice, but the damage especially on photoreceptors was more pronounced in retinae of P2Y1R-KO mice. In contrast, gene expression profiling and histological data suggest an increased survival of amacrine cells in the postischemic retina of P2Y1R-KO mice. Interestingly, measuring the ischemia-induced downregulation of inwardly rectifying potassium channel (Kir)-mediated K⁺ currents as an indicator, reactive Müller cell gliosis was found to be weaker in P2Y1R-KO (current amplitude decreased by 18%) than in Wt mice (decrease by 68%). The inner retina harbors those neurons generating action potentials, which strongly rely on an intact ion homeostasis. This may explain why especially these cells appear to benefit from the preserved Kir4.1 expression in Müller cells, which should allow them to keep up their function in the context of spatial buffering of potassium. Especially under ischemic conditions, maintenance of this Müller cell function may dampen cytotoxic neuronal hyperexcitation and subsequent neuronal cell loss. In sum, we found that purinergic signaling modulates the gliotic activation pattern of Müller glia and lack of P2Y₁ has janus-faced effects. In the end, the differential effects of a disrupted P2Y₁ signaling onto neuronal survival in the ischemic retina call the putative therapeutical use of P2Y₁-antagonists into question.Glial cells are crucially involved in the maintenance of neuronal activity in nervous tissues.¹ The homeostasis of the extracellular space is regulated by various glial functions including spatial K⁺ buffering, cell volume regulation and uptake of neurotransmitters.^{2, 3, 4} Activation of membrane receptors and ion channels is critically implicated in mediating the neuron-supportive glial functions. The dominant K⁺ conductance of glial cells mediates spatial K⁺ buffering and is important for the very negative membrane potential of these cells, thereby supporting electrogenic membrane transporters.⁵ Alterations in glial function are characteristic for pathological processes of the nervous system.⁶ Reactive gliosis may have beneficial and detrimental effects and is considered as an attempt to maintain neuronal function, protecting the tissue from further destruction, and to initiate tissue regeneration.^{7, 8} However, reactive gliosis may cause secondary neuronal damage as major neuron-supportive functions of glial cells get lost.⁶Gliotic alterations of Müller cells, the dominant macroglia of the vertebrate retina, have been observed in various models of retinal diseases.^{9, 10} A prominent feature of Müller cell gliosis is the downregulation of the inwardly rectifying K⁺ conductance mediated by inwardly rectifying K⁺ (Kir) channels.⁹ It has been demonstrated in astrocytes that downregulation or conditional knockout of Kir4.1 results in an impairment of glial glutamate (Glu) uptake.^{11, 12} In addition, it has been suggested that autocrine/paracrine purinergic signaling may have a causative role in the development of reactive gliosis in brain and retina.^{13, 14} Müller cells express different subtypes of P2 nucleotide receptors including P2Y₁ and P2Y₄.^{15, 16} P2Y₁ receptors have been demonstrated to be functionally expressed by Müller cells and microglial cells, rather than by neurons.^{15, 16, 17, 18}Retinal ischemia, a characteristic of various important human blinding diseases including diabetic retinopathy, results in neuronal degeneration and reactive gliosis.^{19, 20} The reduced K⁺ permeability of Müller cell membranes is associated with an impaired cell volume regulation under hypoosmotic stress after high intraocular pressure (HIOP)-induced ischemia.²¹ It has been observed that tandem-pore domain K⁺ channels may fulfill certain functions under conditions where Kir channels are downregulated or lacking.^{22, 23} A malfunctional Müller cell volume regulation was also found after deletion of P2Y₁ in the mouse retina.¹⁶ It has been suggested that impaired glial K⁺ buffering and cell volume regulation may contribute to neuronal degeneration in the ischemic retina by inducing neuronal hyperexcitation and Glu-induced cell death.¹⁴ In order to determine whether endogenous purinergic signaling is implicated in mediating and/or protecting from neuronal degeneration, we investigated the effects of HIOP-induced ischemia in the retinae of P2Y₁-deficient mice.     

The zinc sensing receptor,ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn²⁺ are not well understood. Here, we determined a role of the colonocytic Zn²⁺ sensing receptor, ZnR/GPR39, in mediating Zn²⁺-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn²⁺-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn²⁺ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.The intestinal epithelial barrier, located at the interface between the body and the digestive system lumen, facilitates electrolyte and nutrient absorption while protecting against permeation of antigenic, toxic or infectious materials.¹ Stressful conditions in the intestinal lumen require rapid and continuous renewal of the epithelial layer,² which occur through tightly regulated and balanced proliferation, migration and differentiation processes.³Zn²⁺ deficiency leads to a reduction of epithelial cell proliferation rate, thus limiting renewal of gastrointestinal mucosa, but it also impairs barrier function, increases permeability and enhances cell death.^{4, 5} Zn²⁺ supplementation reverses these processes and restores integrity of colon epithelium.^{5, 6, 7} Importantly, Zn²⁺ supplementation reduces diarrheal disease activity index^{8, 9} and is effective in decreasing severity of histological and clinical scores in in vivo models of ulcerative colitis.^{10, 11} Despite these observations, the signaling pathways linking Zn²⁺ to intestinal epithelial function or integrity are poorly understood.We identified a Zn²⁺ sensing receptor, ZnR, which links between changes in extracellular Zn²⁺ and major intracellular signaling pathways. Functional studies indicated that ZnR is a Gq-coupled receptor, which triggers inositol 1,4,5-trisphosphate (IP3)-dependent release of intracellular Ca²⁺,¹² leading to activation of mitogen-activated protein (MAP) and phosphoinositide 3 (PI3) kinase pathways.^{13, 14} Recent studies showed that the Zn²⁺-dependent Ca²⁺ rise is mediated by the G-protein-coupled receptor GPR39, in colonocytes.¹⁵ ZnR/GPR39 mediates recovery from acidic pH by upregulation of Na⁺/H⁺ exchange in colonocytic and keratinocytic cell lines as well as in native colon epithelial cells, demonstrating the important role of this receptor in epithelial physiology.^{13, 15, 16} Finally, ZnR/GPR39 enhances colonocytes survival from butyrate induced stress.¹⁵ Notably, GPR39 knockout (KO) mice show symptoms of Zn²⁺ deficiency: accelerated gastric emptying and increased fecal secretion.¹⁷ However how Zn²⁺ or ZnR/GPR39 signaling promote colon epithelial function is not well understood. Here we show that ZnR/GPR39 regulates extracellular Zn²⁺-dependent proliferation and differentiation processes in colonocytes. Furthermore, we show that ZnR/GPR39 is essential for expression and localization of tight junction proteins in vitro and in vivo, and thereby regulates the formation of the colon epithelial barrier.     

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.     

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.     

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.     

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.     

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.     

Scaffolding protein Homer1a protects against NMDA-induced neuronal injury

Excessive N-methyl-D-aspartate receptor (NMDAR) activation and the resulting activation of neuronal nitric oxide synthase (nNOS) cause neuronal injury. Homer1b/c facilitates NMDAR-PSD95-nNOS complex interactions, and Homer1a is a negative competitor of Homer1b/c. We report that Homer1a was both upregulated by and protected against NMDA-induced neuronal injury in vitro and in vivo. The neuroprotective activity of Homer1a was associated with NMDA-induced Ca²⁺ influx, oxidative stress and the resultant downstream signaling activation. Additionally, we found that Homer1a functionally regulated NMDAR channel properties in neurons, but did not regulate recombinant NR1/NR2B receptors in HEK293 cells. Furthermore, we found that Homer1a detached the physical links among NR2B, PSD95 and nNOS and reduced the membrane distribution of NMDAR. NMDA-induced neuronal injury was more severe in Homer1a homozygous knockout mice (KO, Homer1a^−/−) when compared with NMDA-induced neuronal injury in wild-type mice (WT, Homer1a^+/+). Additionally, Homer1a overexpression in the cortex of Homer1a^−/− mice alleviated NMDA-induced neuronal injury. These findings suggest that Homer1a may be a key neuroprotective endogenous molecule that protects against NMDA-induced neuronal injury by disassembling NR2B-PSD95-nNOS complexes and reducing the membrane distribution of NMDARs.Glutamate (Glu) acts on glutamate receptors, such as the N-methyl-D-aspartate receptor (NMDAR), and leads to neuronal hyper-excitability and death in a dose-dependent manner.¹ NMDAR activation induces Ca²⁺ influx and specifically activates neuronal nitric oxide synthase (nNOS) and downstream signaling pathways.^{2, 3, 4} Ca²⁺ influx is involved in glutamate-induced apoptosis caused by the activation of apoptosis-related signaling pathways, mitochondrial dysfunction and ROS induction.^{3, 4} Additionally, nNOS has been reported to contribute to NMDA-induced excitotoxicity.^{5, 6} Considering that direct NMDAR inhibition has not yet demonstrated favorable efficacy in most clinic trails and further considering the remarkable role of nNOS in NMDA-induced neuronal death,⁷ measures that can effectively protect neurons from NMDA-induced neuronal injury are urgently needed and represent a worthwhile research goal.Homer proteins belong to the postsynaptic density (PSD) family and consist of two major groups: the short-form Homer proteins (Homer1a and Ania3) and the long-form Homer proteins (Homer1b/c, Homer2 and Homer3).⁸ Homer1b/c has a conserved N-terminal Ena/VASP homology 1 domain and binds to group I metabotropic glutamate receptors (mGluRs), inositol triphosphate receptors and Shank family proteins.^{9, 10, 11, 12} Homer1b/c regulates surface receptor expression,^{13, 14} clustering,¹⁵ transient receptor potential family channels and mGluRs coupled to ion channels.^{10, 16, 17, 18, 19} Additionally, because of its C-terminal coiled-coil (CC) domains, Homer1b/c can self-multimerize, form multiprotein complexes and facilitate signal transduction to downstream pathways. Homer1a, which lacks the CC domain, is believed to compete with constitutive Homer1b/c and disrupt the association of multiple Homer1b/c complexes.Notably, Homer1b/c can interact with the Glu-induced Ca²⁺ influx pathway by binding to Shank, a NMDAR complex adaptor protein (NMDAR-PSD95-GKAP-Shank-Homer1b/c).^{12, 20} Furthermore, Homer1a also interacts with Shank, NMDA, nNOS and other Homer1b/c target proteins. Homer1a has a negative regulatory role by physically replacing certain target proteins, and is involved in the regulation of a variety of cellular and molecular functions in neurological diseases.^{21, 22, 23, 24, 25} Nevertheless, the mechanisms of action and associations between Homer1a and NMDA-induced neuronal injury have not yet been studied. Here, we aimed to investigate the possible neuroprotective effects of Homer1a and explore the mechanisms underlying Homer1a activity in NMDA-induced neuronal injury.