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.     

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.     

Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.The ABCDE model predicts the formation of any flower organ by the interaction of five classes of homeotic genes in plants (Yanofsky et al., 1990; Jack et al., 1992; Mandel et al., 1992; Goto and Meyerowitz, 1994; Jofuku et al., 1994; Pelaz et al., 2000, 2001; Theißen and Saedler, 2001; Pinyopich et al., 2003; Ditta et al., 2004; Jack, 2004). The A class genes control sepal formation. The A, B, and E class genes work together to regulate petal formation. The B, C, and E class genes control stamen formation. The C and E class genes work to regulate carpel formation, whereas the D class gene is involved in ovule development. MADS box genes seem to have a central role in flower development, because most ABCDE genes encode MADS box proteins (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994; Purugganan et al., 1995; Rounsley et al., 1995; Theißen and Saedler, 1995; Theißen et al., 2000; Theißen, 2001).The function of B group genes, such as APETALA3 (AP3) and PISTILLATA (PI), has been thought to have a major role in specifying petal and stamen development (Jack et al., 1992; Goto and Meyerowitz, 1994; Krizek and Meyerowitz, 1996; Kramer et al., 1998; Hernandez-Hernandez et al., 2007; Kanno et al., 2007; Whipple et al., 2007; Irish, 2009). In Arabidopsis (Arabidopsis thaliana), mutation in AP3 or PI caused identical phenotypes of second whorl petal conversion into a sepal structure and third flower whorl stamen into a carpel structure (Bowman et al., 1989; Jack et al., 1992; Goto and Meyerowitz, 1994). Similar homeotic conversions for petal and stamen were observed in the mutants of the AP3 and PI orthologs from a number of core eudicots such as Antirrhinum majus, Petunia hybrida, Gerbera hybrida, Solanum lycopersicum, and Nicotiana benthamiana (Sommer et al., 1990; Tröbner et al., 1992; Angenent et al., 1993; van der Krol et al., 1993; Yu et al., 1999; Liu et al., 2004; Vandenbussche et al., 2004; de Martino et al., 2006), from basal eudicot species such as Papaver somniferum and Aquilegia vulgaris (Drea et al., 2007; Kramer et al., 2007), as well as from monocot species such as Zea mays and Oryza sativa (Ambrose et al., 2000; Nagasawa et al., 2003; Prasad and Vijayraghavan, 2003; Yadav et al., 2007; Yao et al., 2008). This indicated that the function of the B class genes AP3 and PI is highly conserved during evolution.It has been thought that B group genes may have arisen from an ancestral gene through multiple gene duplication events (Doyle, 1994; Theißen et al., 1996, 2000; Purugganan, 1997; Kramer et al., 1998; Kramer and Irish, 1999; Lamb and Irish, 2003; Kim et al., 2004; Stellari et al., 2004; Zahn et al., 2005; Hernandez-Hernandez et al., 2007). In the gymnosperms, there was a single putative B class lineage that duplicated to generate the paleoAP3 and PI lineages in angiosperms (Kramer et al., 1998; Theißen et al., 2000; Irish, 2009). The paleoAP3 lineage is composed of AP3 orthologs identified in lower eudicots, magnolid dicots, and monocots (Kramer et al., 1998). Genes in this lineage contain the conserved paleoAP3- and PI-derived motifs in the C-terminal end of the proteins, which have been thought to be characteristics of the B class ancestral gene (Kramer et al., 1998; Tzeng and Yang, 2001; Hsu and Yang, 2002). The PI lineage is composed of PI orthologs that contain a highly conserved PI motif identified in most plant species (Kramer et al., 1998). Subsequently, there was a second duplication at the base of the core eudicots that produced the euAP3 and TM6 lineages, which have been subject to substantial sequence changes in eudicots during evolution (Kramer et al., 1998; Kramer and Irish, 1999). The paleoAP3 motif in the C-terminal end of the proteins was retained in the TM6 lineage and replaced by a conserved euAP3 motif in the euAP3 lineage of most eudicot species (Kramer et al., 1998). In addition, many lineage-specific duplications for paleoAP3 lineage have occurred in plants such as orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009), Ranunculaceae, and Ranunculales (Kramer et al., 2003; Di Stilio et al., 2005; Shan et al., 2006; Kramer, 2009).Unlike the A or C class MADS box proteins, which form homodimers that regulate flower development, the ability of B class proteins to form homodimers has only been reported in gymnosperms and in the paleoAP3 and PI lineages of some monocots. For example, LMADS1 of the lily Lilium longiflorum (Tzeng and Yang, 2001), OMADS3 of the orchid Oncidium Gower Ramsey (Hsu and Yang, 2002), and PeMADS4 of the orchid Phalaenopsis equestris (Tsai et al., 2004) in the paleoAP3 lineage, LRGLOA and LRGLOB of the lily Lilium regale (Winter et al., 2002), TGGLO of the tulip Tulipa gesneriana (Kanno et al., 2003), and PeMADS6 of the orchid P. equestris (Tsai et al., 2005) in the PI lineage, and GGM2 of the gymnosperm Gnetum gnemon (Winter et al., 1999) were able to form homodimers that regulate flower development. Proteins in the euAP3 lineage and in most paleoAP3 lineages were not able to form homodimers and had to interact with PI to form heterodimers in order to regulate petal and stamen development in various plant species (Schwarz-Sommer et al., 1992; Tröbner et al., 1992; Riechmann et al., 1996; Moon et al., 1999; Winter et al., 2002; Kanno et al., 2003; Vandenbussche et al., 2004; Yao et al., 2008). In addition to forming dimers, AP3 and PI were able to interact with other MADS box proteins, such as SEPALLATA1 (SEP1), SEP2, and SEP3, to regulate petal and stamen development (Pelaz et al., 2000; Honma and Goto, 2001; Theißen and Saedler, 2001; Castillejo et al., 2005).Orchids are among the most important plants in the flower market around the world, and research on MADS box genes has been reported for several species of orchids during the past few years (Lu et al., 1993, 2007; Yu and Goh, 2000; Hsu and Yang, 2002; Yu et al., 2002; Hsu et al., 2003; Tsai et al., 2004, 2008; Xu et al., 2006; Guo et al., 2007; Kim et al., 2007; Chang et al., 2009). Unlike the flowers in eudicots, the nearly identical shape of the sepals and petals as well as the production of a unique lip in orchid flowers make them a very special plant species for the study of flower development. Four clades (1–4) of genes in the paleoAP3 lineage have been identified in several orchids (Hsu and Yang, 2002; Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009; Mondragón-Palomino et al., 2009). Several works have described the possible interactions among these four clades of paleoAP3 genes and one PI gene that are involved in regulating the differentiation and formation of the sepal/petal/lip of orchids (Tsai et al., 2004; Kim et al., 2007; Mondragón-Palomino and Theißen, 2008, 2009). However, the exact mechanism that involves the orchid B class genes remains unclear and needs to be clarified by more experimental investigations.O. Gower Ramsey is a popular orchid with important economic value in cut flower markets. Only a few studies have been reported on the role of MADS box genes in regulating flower formation in this plant species (Hsu and Yang, 2002; Hsu et al., 2003; Chang et al., 2009). An AP3-like MADS gene that regulates both floral formation and initiation in transgenic Arabidopsis has been reported (Hsu and Yang, 2002). In addition, four AP1/AGAMOUS-LIKE9 (AGL9)-like MADS box genes have been characterized that show novel expression patterns and cause different effects on floral transition and formation in Arabidopsis (Hsu et al., 2003; Chang et al., 2009). Compared with other orchids, the production of a large and well-expanded lip and five small identical sepals/petals makes O. Gower Ramsey a special case for the study of the diverse functions of B class MADS box genes during evolution. Therefore, the isolation of more B class MADS box genes and further study of their roles in the regulation of perianth (sepal/petal/lip) formation during O. Gower Ramsey flower development are necessary. In addition to the clade 2 paleoAP3 gene OMADS3, which was previously characterized in our laboratory (Hsu and Yang, 2002), three more B class MADS box genes, OMADS5, OMADS8, and OMADS9, were characterized from O. Gower Ramsey in this study. Based on the different expression patterns and the protein interactions among these four orchid B class genes, we propose that the presence of OMADS3/8 and/or OMADS9 is required for sepal/petal/lip formation. Further sepal and petal formation at least requires the additional presence of OMADS5, whereas large lip formation was seen when OMADS5 expression was absent. Our results provide a new finding and information pertaining to the roles for orchid B class MADS box genes in the regulation of sepal/petal/lip formation.     

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.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Calcineurin B-Like Protein-Interacting Protein Kinase CIPK21 Regulates Osmotic and Salt Stress Responses in Arabidopsis

The role of calcium-mediated signaling has been extensively studied in plant responses to abiotic stress signals. Calcineurin B-like proteins (CBLs) and CBL-interacting protein kinases (CIPKs) constitute a complex signaling network acting in diverse plant stress responses. Osmotic stress imposed by soil salinity and drought is a major abiotic stress that impedes plant growth and development and involves calcium-signaling processes. In this study, we report the functional analysis of CIPK21, an Arabidopsis (Arabidopsis thaliana) CBL-interacting protein kinase, ubiquitously expressed in plant tissues and up-regulated under multiple abiotic stress conditions. The growth of a loss-of-function mutant of CIPK21, cipk21, was hypersensitive to high salt and osmotic stress conditions. The calcium sensors CBL2 and CBL3 were found to physically interact with CIPK21 and target this kinase to the tonoplast. Moreover, preferential localization of CIPK21 to the tonoplast was detected under salt stress condition when coexpressed with CBL2 or CBL3. These findings suggest that CIPK21 mediates responses to salt stress condition in Arabidopsis, at least in part, by regulating ion and water homeostasis across the vacuolar membranes.Drought and salinity cause osmotic stress in plants and severely affect crop productivity throughout the world. Plants respond to osmotic stress by changing a number of cellular processes (Xiong et al., 1999; Xiong and Zhu, 2002; Bartels and Sunkar, 2005; Boudsocq and Lauriére, 2005). Some of these changes include activation of stress-responsive genes, regulation of membrane transport at both plasma membrane (PM) and vacuolar membrane (tonoplast) to maintain water and ionic homeostasis, and metabolic changes to produce compatible osmolytes such as Pro (Stewart and Lee, 1974; Krasensky and Jonak, 2012). It has been well established that a specific calcium (Ca²⁺) signature is generated in response to a particular environmental stimulus (Trewavas and Malhó, 1998; Scrase-Field and Knight, 2003; Luan, 2009; Kudla et al., 2010). The Ca²⁺ changes are primarily perceived by several Ca²⁺ sensors such as calmodulin (Reddy, 2001; Luan et al., 2002), Ca²⁺-dependent protein kinases (Harper and Harmon, 2005), calcineurin B-like proteins (CBLs; Luan et al., 2002; Batistič and Kudla, 2004; Pandey, 2008; Luan, 2009; Sanyal et al., 2015), and other Ca²⁺-binding proteins (Reddy, 2001; Shao et al., 2008) to initiate various cellular responses.Plant CBL-type Ca²⁺ sensors interact with and activate CBL-interacting protein kinases (CIPKs) that phosphorylate downstream components to transduce Ca²⁺ signals (Liu et al., 2000; Luan et al., 2002; Batistič and Kudla, 2004; Luan, 2009). In several plant species, multiple members have been identified in the CBL and CIPK family (Luan et al., 2002; Kolukisaoglu et al., 2004; Pandey, 2008; Batistič and Kudla, 2009; Weinl and Kudla, 2009; Pandey et al., 2014). Involvement of specific CBL-CIPK pair to decode a particular type of signal entails the alternative and selective complex formation leading to stimulus-response coupling (D’Angelo et al., 2006; Batistič et al., 2010).Several CBL and CIPK family members have been implicated in plant responses to drought, salinity, and osmotic stress based on genetic analysis of Arabidopsis (Arabidopsis thaliana) mutants (Zhu, 2002; Cheong et al., 2003, 2007; Kim et al., 2003; Pandey et al., 2004, 2008; D’Angelo et al., 2006; Qin et al., 2008; Tripathi et al., 2009; Held et al., 2011; Tang et al., 2012; Drerup et al., 2013; Eckert et al., 2014). A few CIPKs have also been functionally characterized by gain-of-function approach in crop plants such as rice (Oryza sativa), pea (Pisum sativum), and maize (Zea mays) and were found to be involved in osmotic stress responses (Mahajan et al., 2006; Xiang et al., 2007; Yang et al., 2008; Tripathi et al., 2009; Zhao et al., 2009; Cuéllar et al., 2010).In this report, we examined the role of the Arabidopsis CIPK21 gene in osmotic stress response by reverse genetic analysis. The loss-of-function mutant plants became hypersensitive to salt and mannitol stress conditions, suggesting that CIPK21 is involved in the regulation of osmotic stress response in Arabidopsis. These findings are further supported by an enhanced tonoplast targeting of the cytoplasmic CIPK21 through interaction with the vacuolar Ca²⁺ sensors CBL2 and CBL3 under salt stress condition.     

Naturally Occurring Disseminated Group B Streptococcus Infections in Postnatal Rats

Group B Streptococcus (Streptococcus agalactiae, GBS) is a gram-positive commensal and occasional opportunistic pathogen of the human vaginal, respiratory, and intestinal tracts that can cause sepsis, pneumonia, or meningitis in human neonates, infants, and immunosuppressed persons. We report here on a spontaneous outbreak of postnatal GBS-associated disease in rats. Ten of 26 (38.5%) 21- to 24-d-old rat pups died or were euthanized due to a moribund state in a colony of rats transgenic for the human diphtheria toxin receptor on a Munich–Wistar–Frömter genetic background. Four pups had intralesional coccoid bacteria in various organs without accompanying inflammation. GBS was isolated from the liver of 2 of these pups and from skin abscesses in 3 littermates. A connection with the transgene could not be established. A treatment protocol was evaluated in the remaining breeding female rats. GBS is a potentially clinically significant spontaneous infection in various populations of research rats, with some features that resemble late-onset postnatal GBS infection in human infants.Abbreviations: GBS, Group B Streptococcus; MWF, Munich Wistar Frömter; hDTR, human diphtheria toxin receptorStreptococci are gram-positive, coccoid bacteria that typically are classified according to their hemolytic capacity. α-hemolytic streptococci produce a zone of partial hemolysis that appears greenish on blood agar, whereas β-hemolytic streptococci produce a zone of complete hemolysis, and γ-hemolytic organisms produce no hemolysis on blood agar.²⁴ The β-hemolytic streptococci are further subdivided into Lancefield groups (A through G), according to cell-wall carbohydrate antigens.^24,29,39 The group B β-hemolytic Streptococcus (GBS) have been speciated as Streptococcus agalactiae.^28,39 It was first isolated as a causative agent of mastitis in cattle.²⁹ This organism has since been recognized as a cause of severe infection in human neonates.^28,39 In humans, GBS is harbored asymptomatically in the maternal genitourinary tract.^24,28 Infants can be infected and present with serious systemic disease in the first week of life (early-onset GBS) or from 1 wk to 3 mo of age (late-onset GBS).³⁹ In laboratory animals, rats have been used experimentally as models for neonatal^{1,6,7,20,37,38,43,44,47,50,51} or adult⁴⁵ GBS infection, but to our knowledge, GBS has not been associated with spontaneous disease in rats.     

Alzheimer's disease-related amyloid-β induces synaptotoxicity in human iPS cell-derived neurons

Human induced pluripotent stem cell (iPSC)-derived neurons have been proposed to be a highly valuable cellular model for studying the pathomechanisms of Alzheimer''s disease (AD). Studies employing patient-specific human iPSCs as models of familial and sporadic forms of AD described elevated levels of AD-related amyloid-β (Aβ). However, none of the present AD iPSC studies could recapitulate the synaptotoxic actions of Aβ, which are crucial early events in a cascade that eventually leads to vast brain degeneration. Here we established highly reproducible, human iPSC-derived cortical cultures as a cellular model to study the synaptotoxic effects of Aβ. We developed a highly efficient immunopurification procedure yielding immature neurons that express markers of deep layer cortical pyramidal neurons and GABAergic interneurons. Upon long-term cultivation, purified cells differentiated into mature neurons exhibiting the generation of action potentials and excitatory glutamatergic and inhibitory GABAergic synapses. Most interestingly, these iPSC-derived human neurons were strongly susceptible to the synaptotoxic actions of Aβ. Application of Aβ for 8 days led to a reduction in the overall FM4–64 and vGlut1 staining of vesicles in neurites, indicating a loss of vesicle clusters. A selective analysis of presynaptic vesicle clusters on dendrites did not reveal a significant change, thus suggesting that Aβ impaired axonal vesicle clusters. In addition, electrophysiological patch-clamp recordings of AMPA receptor-mediated miniature EPSCs revealed an Aβ-induced reduction in amplitudes, indicating an impairment of postsynaptic AMPA receptors. A loss of postsynaptic AMPA receptor clusters was confirmed by immunocytochemical stainings for GluA1. Incubation with Aβ for 8 days did not result in a significant loss of neurites or cell death. In summary, we describe a highly reproducible cellular AD model based on human iPSC-derived cortical neurons that enables the mechanistic analysis of Aβ-induced synaptic pathomechanisms and the development of novel therapeutic approaches.In Alzheimer''s disease (AD), synapse damage and synapse loss are thought to underlie cognitive deficits.¹ Oligomers of the amyloid-β (Aβ) peptide appear to induce synaptic failure as an early event in the etiology of AD.^{2, 3, 4} However, despite its well-established synapse-impairing effects in rodent models,^{5, 6, 7} the synaptotoxic actions of Aβ most relevant for the human disease have not been identified in a human model system. Several studies have investigated the synaptotoxic effects of Aβ in cultured rodent neurons and in transgenic mouse models revealing a multitude of potential mechanisms affecting synapses. Postsynaptic Aβ actions result in the loss of functional (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type) glutamate receptors,^{8, 9, 10} involve long-term depression-like mechanisms,^{9, 11, 12} and lead to the degradation of the entire postsynapse (dendritic spines).^{9, 11, 13} In addition, several distinct presynaptic Aβ actions on the synaptic vesicle cycle have been described.^{10, 14} Furthermore, Aβ-induced impairments of axonal transport regulation and Aβ-induced axon degeneration have been found in rodent neurons.^{15, 16, 17} This puzzling diversity of Aβ-induced synapse-related defects raises the question whether all of them are involved in the early pathomechanisms of human AD.In addition to well-established animal systems, the modelling of human neurological disease pathologies by human induced pluripotent stem cell (hiPSC) technology¹⁸ has been proposed as an innovative approach.^{19, 20, 21} The in vitro differentiation of hiPSCs to excitable neurons has been reported using a variety of protocols.^{22, 23, 24} However, quantitative analysis of both functional glutamatergic and GABAergic synapses has been difficult to achieve.^{19, 25, 26} In addition to studying the functional properties of iPSC-derived human neurons from healthy individuals, the in vitro differentiation of patient-derived iPSCs has been used to model complex neurodevelopmental and neurodegenerative diseases.^{19, 27, 28} Recently, iPSCs derived from AD patients have been reported to exhibit increased secretion of Aβ upon in vitro neuronal differentiation; however, neither a loss of synapses nor an impairment of synapse function was detected.^{21, 29, 30, 31, 32, 33} Here we describe a hiPSC-based, carefully optimized in vitro differentiation protocol, including a novel immunopanning step, which enabled us to study the deleterious effects of application of Aβ on human cortical neurons and on human synapses.     

The K-Segment of Maize DHN1 Mediates Binding to Anionic Phospholipid Vesicles and Concomitant Structural Changes

Dehydrins (DHNs; late embryogenesis abundant D11 family) are a family of intrinsically unstructured plant proteins that accumulate in the late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid treatment. We demonstrated previously that maize (Zea mays) DHNs bind preferentially to anionic phospholipid vesicles; this binding is accompanied by an increase in α-helicity of the protein, and adoption of α-helicity can be induced by sodium dodecyl sulfate. All DHNs contain at least one “K-segment,” a lysine-rich 15-amino acid consensus sequence. The K-segment is predicted to form a class A2 amphipathic α-helix, a structural element known to interact with membranes and proteins. Here, three K-segment deletion proteins of maize DHN1 were produced. Lipid vesicle-binding assays revealed that the K-segment is required for binding to anionic phospholipid vesicles, and adoption of α-helicity of the K-segment accounts for most of the conformational change of DHNs upon binding to anionic phospholipid vesicles or sodium dodecyl sulfate. The adoption of structure may help stabilize cellular components, including membranes, under stress conditions.When plants encounter environmental stresses such as drought or low temperature, various responses take place to adapt to these conditions. Typical responses include increased expression of chaperones, signal transduction pathway and late embryogenesis abundant (LEA) proteins, osmotic adjustment, and induction of degradation and repair systems (Ingram and Bartels, 1996).Dehydrins (DHNs; LEA D11 family) are a subfamily of group 2 LEA proteins that accumulate to high levels during late stages of seed development and in vegetative tissues subjected to water deficit, salinity, low temperature, or abscisic acid (ABA) treatment (Svensson et al., 2002). Some DHNs are expressed constitutively during normal growth (Nylander et al., 2001; Rorat et al., 2004, 2006; Rodriguez et al., 2005). DHNs exist in a wide range of photosynthetic organisms, including angiosperms, gymnosperms, algae, and mosses (Svensson et al., 2002). DHNs are encoded by a dispersed multigene family and are differentially regulated, at least in higher plants. For example, 13 Dhn genes have been identified in barley (Hordeum vulgare), dispersed over seven genetic map locations (Choi et al., 1999; Svensson et al., 2002) and regulated variably by drought, low temperature, and embryo development (Tommasini et al., 2008). DHNs are localized in various subcellular compartments, including cytosol (Roberts et al., 1993), nucleus (Houde et al., 1995), chloroplast (Artus et al., 1996), vacuole (Heyen et al., 2002), and proximal to the plasma membrane and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Puhakainen et al., 2004). Elevated expression of Dhn genes generally has been correlated with the acquisition of tolerance to abiotic stresses such as drought (Whitsitt et al., 1997), salt (Godoy et al., 1994; Jayaprakash et al., 1998), chilling (Ismail et al., 1999a), or freezing (Houde et al., 1995; Danyluk et al., 1998; Fowler et al., 2001). The differences in expression and tissue location suggest that individual members of the Dhn multigene family have somewhat distinct biological functions (Close, 1997; Zhu et al., 2000; Nylander et al., 2001). Many studies have observed a positive correlation between the accumulation of DHNs and tolerance to abiotic stresses (Svensson et al., 2002). However, overexpression of a single DHN protein has not, in general, been sufficient to confer stress tolerance (Puhakainen et al., 2004).DHNs are subclassified by sequence motifs referred to as the K-segment (Lys-rich consensus sequence), the Y-segment (N-terminal conserved sequence), the S-segment (a tract of Ser residues), and the φ-segment (Close, 1996). Because of high hydrophilicity, high content of Gly (>20%), and the lack of a defined three-dimensional structure in the pure form (Lisse et al., 1996), DHNs have been categorized as “intrinsically disordered/unstructured proteins” or “hydrophilins” (Wright and Dyson, 1999; Garay-Arroyo et al., 2000; Tompa, 2005; Kovacs et al., 2008). On the basis of compositional and biophysical properties and their link to abiotic stresses, several functions of DHNs have been proposed, including ion sequestration (Roberts et al., 1993), water retention (McCubbin et al., 1985), and stabilization of membranes or proteins (Close, 1996, 1997). Observations from in vitro experiments include DHN binding to lipid vesicles (Koag et al., 2003; Kovacs et al., 2008) or metals (Svensson et al., 2000; Heyen et al., 2002; Kruger et al., 2002; Alsheikh et al., 2003; Hara et al., 2005), protection of membrane lipid against peroxidation (Hara et al., 2003), retention of hydration or ion sequestration (Bokor et al., 2005; Tompa et al., 2006), and chaperone activity against the heat-induced inactivation and aggregation of various proteins (Kovacs et al., 2008).Intrinsically disordered/unstructured proteins that lack a well-defined three-dimensional structure have recently been recognized to be prevalent in prokaryotes and eukaryotes (Oldfield et al., 2005). They fulfill important functions in signal transduction, gene expression, and binding to targets such as protein, RNA, ions, and membranes (Wright and Dyson, 1999; Tompa, 2002; Dyson and Wright, 2005). The disorder confers structural flexibility and malleability to adapt to changes in the protein environment, including water potential, pH, ionic strength, and temperature, and to undergo structural transition when complexed with ligands such as other proteins, DNA, RNA, or membranes (Prestrelski et al., 1993; Uversky, 2002). Structural changes from disorder to ordered functional structure also can be induced by the folding of a partner protein (Wright and Dyson, 1999; Tompa, 2002; Mouillon et al., 2008).The idea that DHNs interact with membranes is consistent with many immunolocalization studies, which have shown that DHNs accumulate near the plasma membrane or membrane-rich areas surrounding lipid and protein bodies (Asghar et al., 1994; Egerton-Warburton et al., 1997; Danyluk et al., 1998; Puhakainen et al., 2004). The K-segment is predicted to form a class A2 amphipathic α-helix, in which hydrophilic and hydrophobic residues are arranged on opposite faces (Close, 1996). The amphipathic α-helix is a structural element known to interact with membranes and proteins (Epand et al., 1995). Also, in the presence of helical inducers such as SDS and trifluoroethanol (Dalal and Pio, 2006), DHNs take on α-helicity (Lisse et al., 1996; Ismail et al., 1999b). We previously examined the binding of DHN1 to liposomes and found that DHNs bind preferentially to anionic phospholipids and that this binding is accompanied by an increase in α-helicity of the protein (Koag et al., 2003). Similarly, a mitochondrial LEA protein, one of the group III LEA proteins, recently has been shown to interact with and protect membranes subjected to desiccation, coupled with the adoption of amphipathic α-helices (Tolleter et al., 2007).Here, we explore the basis of DHN-vesicle interaction using K-segment deletion proteins. This study reveals that the K-segment is necessary and sufficient for binding to anionic phospholipid vesicles and that the adoption of α-helicity of DHN proteins can be attributed mainly to the K-segment.     

Alpha-Synuclein affects neurite morphology,autophagy, vesicle transport and axonal degeneration in CNS neurons

Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (αSyn-WT), a protein associated with PD, and its mutant variants αSyn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of αSyn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of αSyn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with αSyn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all αSyn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by αSyn-WT and -A53T but not by αSyn-A30P. Correspondingly, colocalization of αSyn and the autophagy marker LC3 was reduced for αSyn-A30P compared with the other αSyn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both αSyn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that αSyn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.Growing evidence suggests that Parkinson''s disease (PD) pathology starts at the presynaptic terminals and the distal axons and is then propagated back to the soma in a ''dying back'' pattern.^{1, 2} Accordingly, at the time of clinical onset, there is only a 30% loss of total substantia nigra pars compacta neurons but a far more severe loss of striatal dopaminergic markers (70–80%), suggesting that axonal terminals of the nigrostriatal pathway are affected earlier.¹ It is thus essential to understand the pathomechanisms specifically affecting the axon in PD in order to interfere with early disease progression.Neurodegeneration in PD is accompanied by the appearance of intraneuronal protein aggregates, denoted Lewy bodies (LBs).³ Interestingly, also LB pathology is initially found in the distal axons before becoming evident in the neuronal somata, and dystrophic neurites, so called ''Lewy neurites'', outnumber LBs in the early stages of PD.^{2, 4, 5} A main component of LBs is the protein alpha-synuclein (αSyn) that is not only widely used as a histopathological marker for PD but is also believed to have a major role in PD pathogenesis.^{6, 7} The importance of αSyn is further underlined by the discovery of αSyn point mutations (e.g. Ala53Thr (A53T), Ala30Pro (A30P)) and multiplications of the αSyn gene, all of which cause autosomal dominant forms of PD.^{8, 9, 10} However, neither the physiological functions nor the pathogenetic mechanisms of αSyn are well understood.⁷The biological effects of αSyn expression strongly depend on the model system. Wild-type (WT) human αSyn does not lead to major clinical or histological abnormalities when expressed in transgenic mice,^{11, 12} but its overexpression mediated by adeno-associated viral vectors (AAV) results in severe neurodegeneration, suggesting a dose-dependent toxic effect.^{13, 14} Different human αSyn-A30P and -A53T transgenic mouse lines develop severe motor impairments, partly resembling symptoms of human PD, accompanied by a degeneration of the nigrostriatal neuronal system and LB-like pathology.^{11, 12, 15} In line with the pathological findings in human PD, the axonal compartment is affected early and most prominently in these animal models.Different putative pathomechanisms of αSyn toxicity have been explored. For example, the cytoskeleton is an important molecular target of αSyn. Multimeric forms of αSyn were shown to impair the polymerization of tubulin and microtubule formation.^{16, 17} Overexpression of αSyn increased actin instability and induced actin bundling in cultured hippocampal neurons.¹⁸ There are, however, divergent data on the resulting effects of αSyn overexpression on neurite outgrowth and integrity in different model systems.^{19, 20, 21, 22}Moreover, a dysregulation of autophagy has been implicated in PD pathology. Aberrant αSyn is normally degraded by autophagy and only to a negligible degree by the proteasome.²³ Several studies have shown that the inhibition of autophagy results in an accumulation and increased toxicity of αSyn, whereas the activation of autophagy has therapeutic effects in PD models.^{23, 24, 25, 26} However, the direct effects of αSyn and its mutants on autophagy seem to rely strongly on the model system and the published data are highly controversial.^{24, 26, 27, 28, 29, 30, 31, 32}Given the central role of axonal degeneration in PD, it is likely that disturbances of axonal transport are involved.³³ In support of this proposition, the motor protein kinesin was shown to be decreased early and stage-dependently in PD patients, preceding the loss of substantia nigra neurons.³⁴ αSyn itself is actively transported along the axons, mainly by the slow component of axonal transport, but the role of αSyn in axonal vesicle transport is unclear.³⁵Here, we present a comprehensive analysis of the effects of αSyn on neurite morphology and examine important pathomechanisms.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.     

Neuritin 1 promotes retinal ganglion cell survival and axonal regeneration following optic nerve crush

Neuritin 1 (Nrn1) is an extracellular glycophosphatidylinositol-linked protein that stimulates axonal plasticity, dendritic arborization and synapse maturation in the central nervous system (CNS). The purpose of this study was to evaluate the neuroprotective and axogenic properties of Nrn1 on axotomized retinal ganglion cells (RGCs) in vitro and on the in vivo optic nerve crush (ONC) mouse model. Axotomized cultured RGCs treated with recombinant hNRN1 significantly increased survival of RGCs by 21% (n=6–7, P<0.01) and neurite outgrowth in RGCs by 141% compared to controls (n=15, P<0.05). RGC transduction with AAV2-CAG–hNRN1 prior to ONC promoted RGC survival (450%, n=3–7, P<0.05) and significantly preserved RGC function by 70% until 28 days post crush (dpc) (n=6, P<0.05) compared with the control AAV2-CAG–green fluorescent protein transduction group. Significantly elevated levels of RGC marker, RNA binding protein with multiple splicing (Rbpms; 73%, n=5–8, P<0.001) and growth cone marker, growth-associated protein 43 (Gap43; 36%, n=3, P<0.01) were observed 28 dpc in the retinas of the treatment group compared with the control group. Significant increase in Gap43 (100%, n=5–6, P<0.05) expression was observed within the optic nerves of the AAV2–hNRN1 group compared to controls. In conclusion, Nrn1 exhibited neuroprotective, regenerative effects and preserved RGC function on axotomized RGCs in vitro and after axonal injury in vivo. Nrn1 is a potential therapeutic target for CNS neurodegenerative diseases.Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of intrinsic and extrinsic cellular events resulting in regenerative failure and subsequent damage to neurons.^{1, 2, 3, 4, 5} The intrinsic factors include deregulation in growth-promoting factors, apoptotic factors, intracellular signaling molecules and trophic factors.⁶ Similarly, the extrinsic factors correlate to growth inhibition due to inhibitory cues^{3, 7, 8, 9, 10, 11, 12, 13} that include myelin and myelin associated inhibitors, glial scarring,^{5, 14} slow clearance of axonal debris,⁷ incorrect development of neuronal projections⁶ and CNS inflammation.^{15, 16} Progressive degeneration of mature retinal ganglion cells (RGCs) has been associated with loss of trophic support,^{8, 9} detrimental inflammatory processes/immune regulation^{10, 11} and apoptotic effectors.^{9, 12, 13, 15, 17}After injury, mammalian RGC axons show only a short-lived sprouting response but no long-distance regeneration through the optic nerve (ON).¹⁶ Glial responses around the affected area are initiated by injured CNS axons.¹⁸ Axons undergoing Wallerian degeneration are surrounded by astrocytes that upregulate glial fibrillary acidic protein (Gfap) expression and these reactive astrocytes contribute to trauma-induced neurodegeneration.¹⁹ Glial scarring inhibits axonal transport after ON crush (ONC)^{5, 14} decreasing transport of proteins involved in neuroprotection and synaptic plasticity. Regenerative failure is a critical endpoint of these destructive triggers culminating in neuronal apoptosis^{3, 20, 21} and inhibition of functional recovery. Intrinsic factors affecting axonal regeneration after CNS injury are crucial for recovery and thus, dysregulation of genes involved in axonal plasticity and outgrowth can prove detrimental to the neuronal recovery.^{22, 23, 24}Current neuroprotection approaches include promoting survival of RGCs by intraocular injections of recombinant factors like ciliary neurotrophic factor (CNTF) and peripheral nerve (PN) transplantations in vitro²⁵ and in vivo after injury.²⁶ Studies performed with glial cell-line-derived neurotrophic factor and neurturin protect RGCs from axotomy-induced apoptosis.²⁷ Further, in the ON injury model, RGC survival was promoted after deletion of CCAAT/enhancer binding protein homologous protein²⁸ and enhanced regeneration observed with co-deletion of kruppel-like factor 4 (Klf4) and suppressor of cytokine signaling 3 (Socs3).²⁹ Intraocular administration of neurotrophin-4 (NT-4) and brain-derived neurotrophic factor (BDNF) after ON transection has also exerted neuroprotective effects on axotomized RGCs. In addition, PNs transplanted adjacent to ONs, ex vivo PN grafts with lenti-viral transduced Schwann cells, and stimulation of inflammatory processes have strong pro-regenerative effects on injured RGCs.^{26, 30, 31, 32, 33}In addition, using adeno-associated-virus (AAV) therapy, AAV mediated expression of CNTF in bcl2 overexpressing transgenic mice increases cell viability and axonal regeneration,³⁴ whereas BDNF promotes survival of RGCs.³⁵ Likewise, experiments with AAV–BDNF, –CNTF and –growth-associated protein 43 (GAP43) have shown that AAV–CNTF was the most crucial for promoting both long-term survival and regeneration.³⁶ The positive effects of CNTF are observed mainly through simultaneous deletion of both PTEN and SOCS3³⁷ and the concurrent activation of mTOR and STAT3 pathways.³⁸ Although CNTF shows robust increase and sustained axon regeneration in injured ONs of rodents, it causes axonal misguidance and aberrant growth.³⁹ Furthermore, it has been shown that CNTF acts as a chemoattractant. CNTF administration onto autologous PN grafts transplanted within transected ON increased regeneration, but these effects were significantly reduced after removal of macrophages from this site.⁴⁰ In addition, the effects of CNTF using PN grafts at ON transection sites are further subject to debate, as previously it has been shown that Ad-CNTF injections preserved RGC axons but did not induce regeneration of axotomized RGCs.⁴¹ Thus, other studies have addressed RGC survivability and axonal regeneration with CNTF and other growth factors,^{35, 36} but most trophic factors affect neuronal survival and regeneration differentially.Previous studies targeting neuronal apoptosis by overexpressing intrinsic growth factors, inhibiting apoptosis and enhancing regeneration in CNS trauma models have established that a multifactorial approach is required for successful and long-lasting therapeutic outcomes.^{6, 36} Current gaps still exist for a key gene that could effectively target neuroprotection, enhance neuron regeneration and sustain neuronal function.One key gene implicated in neuronal plasticity is Neuritin 1 (Nrn1), also known as candidate plasticity gene 15. It has multiple functions and was first identified and characterized when screening for candidate plasticity genes in the rat hippocampal dentate gyrus activated by kainate.^{42, 43, 44} Nrn1 is highly conserved across species⁴⁵ and translates to an extracellular, glycophosphatidylinositol-linked protein (GPI-linked protein), which can be secreted as a soluble form. Nrn1 stimulates axonal plasticity, dendritic arborization and synapse maturation in the CNS.⁴⁶ During early embryonic development, Nrn1 promotes the survival of neural progenitors and differentiated neurons,⁴⁷ while later in development it promotes axonal and dendritic growth and stabilization, allowing maturation and formation of synapses.^{43, 46, 48} In the adult brain, Nrn1 has been correlated with activity-dependent functional plasticity^{45, 49} and is expressed in post mitotic neurons.Nrn1 may be a crucial gene for neuroprotection and regeneration because growth factors such as nerve growth factor (NGF), BDNF and NT-3 as well as neuronal activity can potentiate the expression of Nrn1.^{44, 50} In addition, we reported that Nrn1 mRNA expression appears to be biphasic after ON axonal trauma, indicating a transient attempt by RGCs at neuroprotection/neuroregeneration in response to ONC injury.⁵¹ The dynamic regulation of Nrn1 coupled with neurotrophic effects may promote axonal regeneration in the CNS. To overcome CNS trauma, a new therapy geared towards neuroprotection and effective axonal regeneration is required to enhance a future multifactorial approach. The purpose of this study is to evaluate the therapeutic effects of Nrn1 in mouse RGC cultures as well as in the mouse ONC model. We have identified a distinct neuroprotective and regenerative strategy that prevents neurodegeneration after ON injury. AAV2–hNRN1 expression vectors partially rescued RGCs from apoptosis, maintained RGC function, and initiated regeneration of injured axons.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.