A +TIP for a smooth trip

Is there a cellular mechanism for preventing a depolymerizing microtubule track from “slipping out from under” its cargo? A recent study in budding yeast indicates that when a chromosome is transported to the minus end of a spindle microtubule, its kinetochore-bound microtubule plus end–tracking protein (+TIP) Stu2 may move to the plus end to promote rescue; i.e., to switch the depolymerizing end to a polymerizing end. The possibility that other +TIPs may play a similar role in sustaining a microtubule track during vesicular transport deserves investigation.Microtubule motor proteins such as dynein and kinesins are responsible for transporting cellular cargos along microtubule tracks (Vale, 2003). The net direction and speed of cargo movement, however, are likely to be regulated in a very complicated fashion, especially when a cargo is bound to multiple motors with opposite directionalities (Vale 2003; Mallik and Gross 2004; Levi et al., 2006). The fact that the microtubule track is not very stable further complicates matters. The plus ends of microtubules, which face the cell periphery in most cell types, are highly dynamic, exhibiting alternating periods of polymerization (growth) and depolymerization (shrinkage; Desai and Mitchison, 1997). Such plus end dynamics may be useful for searching and capturing relatively stationary cargos near the cell periphery that need to be transported inward (Vaughan et al., 2002). However, the dynamic nature of the track can also create an obvious problem for the transport process. If a microtubule''s rate of shrinkage is greater than the rate of cargo transport, then the microtubule may shrink past an attached cargo, causing its dissociation from the track. Does this happen in cells, or do cells have a mechanism to prevent it?Although this question has never been directly addressed, a recent study on budding yeast chromosome segregation has shed new light on the issue (Tanaka et al., 2005). In this study, the authors took advantage of a strategy that allowed them to specifically shut off the function of a single kinetochore, thereby preventing it from attaching to a spindle microtubule while, at the same time, permitting other kinetochores to attach to the spindle. After the function of this single kinetochore was switched back on, the behavior of its associated chromosome on a spindle microtubule was subjected to a detailed image analysis. Several important insights from this study on chromosome–microtubule interactions during mitosis have been recently reviewed (Bloom 2005), and, thus, only those observations that pertain to cargo transport will be highlighted here. The chromosome was first seen to undergo a lateral interaction with the microtubule followed by minus end–directed transport toward the pole. The mechanism of the minus end–directed transport is not entirely clear, although a member of the kinesin-14 family, Kar3, may be one of the players in this process (Tanaka et al., 2005). During transport, the attached microtubule can undergo shrinkage with a rate higher than that of the minus end–directed chromosome movement (Tanaka et al., 2005); however, it never shrank beyond the position of the cargo. Such exquisite control over the extent of shrinkage appears to rely on a conversation between the cargo and the plus end of the microtubule that is mediated by the microtubule plus end–tracking protein Stu2 (Bloom 2005; Tanaka et al., 2005).Microtubule plus end–tracking proteins (+TIPs) are a class of proteins that use different structural motifs or specific targeting mechanisms to localize to the dynamic plus ends of microtubules (Carvalho et al., 2003; Akhmanova and Hoogenraad, 2005). Although most +TIPs associate with only the growing ends of microtubules, several +TIPs also localize to the shrinking ends (Carvalho et al., 2003; 2004; Akhmanova and Hoogenraad, 2005; Mennella et al., 2005; Sproul et al., 2005; Molk et al., 2006; Wu et al., 2006). Many +TIPs have been found to impact microtubules by either promoting their growth or promoting dynamic behavior. Stu2 is a member of the XMAP215/TOG/Dis1/DdCP224 family of proteins that have been shown to affect microtubule dynamics in multiple ways depending on different experimental conditions (Ohkura et al., 2001; Popov and Karsenti 2003; Holmfeldt et al., 2004; Akhmanova and Hoogenraad, 2005). In vitro, Stu2 binds to the plus ends of preformed microtubules and promotes catastrophe, which is a switch from growth to shrinkage (van Breugel et al., 2003). In vivo studies using mutants of Stu2, however, indicate that Stu2 promotes microtubule growth (Severin et al., 2001) and the dynamics of both kinetochore and cytoplasmic microtubules (Kosco et al., 2001; Pearson et al., 2003). During anaphase B spindle elongation, Stu2 may antagonize the function of Kip3 (a kinesin-13 family member) to promote the plus end polymerization of overlapping microtubules (Severin et al., 2001). Although it is not fully understood how or why Stu2 is so versatile, it is well recognized that the in vivo interactions among +TIPs are very complicated, and the loss of function of a +TIP in vivo may decrease or increase the accumulation of other +TIPs that also regulate microtubule dynamics (Carvalho et al., 2003; 2004; Lansbergen et al., 2004; Akhmanova and Hoogenraad, 2005; Galjart 2005; Komarova et al., 2005). Tanaka et al. (2005) identified Stu2 as a rescue (a switch from shrinkage to growth) factor based not on phenotypic studies of Stu2 mutants but, instead, on a direct observation of the relationship between microtubule plus end behavior and Stu2 localization. They found that Stu2 was localized at the plus ends of microtubules emanating from the spindle pole body, and, during periods of microtubule shrinkage, Stu2 levels at the plus ends were decreased. Interestingly, Stu2 was also localized at the unbound kinetochore. When the kinetochore subsequently attached laterally to a spindle microtubule and underwent minus end–directed transport, the Stu2 proteins were transported from the kinetochore to the microtubule plus end. The arrival of Stu2 at the plus end closely correlated to the rescue of the shrinking microtubule (Tanaka et al., 2005). These observations strongly suggest that the Stu2 carried by the kinetochore may serve as a rescue factor for the microtubule track, preventing it from vanishing before the migrating chromosome.Could such a scenario exist during microtubule-dependent transport of nonchromosomal cargoes during interphase? We do not yet know the answer. However, based on published studies, it seems reasonable to hypothesize that other +TIPs, especially the cytoplasmic linker protein CLIP-170, may function in a manner similar to yeast Stu2 to ensure a safe trip for a minus end–directed cargo. CLIP-170 contains CAP-Gly microtubule-binding motifs at its NH₂ terminus and was initially identified as a protein required for linking endocytic vesicles to microtubules in vitro (Pierre et al., 1992; Rickard and Kreis 1996). Later, CLIP-170 was identified as a founding member of the microtubule plus end–tracking proteins (Perez et al., 1999). The connection between CLIP-170''s in vitro endosome–microtubule linking property and its in vivo plus end tracking behavior has not been clearly made. Could an endocytic vesicle use its bound CLIP-170 as a rescue factor to prevent the disappearance of the track on which it is traveling?CLIP-170 is indeed considered to be a rescue factor in mammalian cells (Komarova et al., 2002a). Komarova et al. (2002b) have found that in cultured CHO and NRK cells, microtubule dynamics seem to be controlled spatially; catastrophe and rescue occur frequently only near the cell periphery. Although the mechanisms behind catastrophe and rescue are not fully understood, protein factors are required for regulating both events in vivo (Desai and Mitchison, 1997). In CHO cells, a dominant-negative form of CLIP-170 that displaces the endogenous CLIP-170 from microtubule plus ends severely reduces the rescue frequency so that microtubules are more likely to shrink all the way back to the microtubule-organizing center (Komarova et al., 2002a). Moreover, both in vivo and in vitro studies suggest that the rescue activity of CLIP-170 is localized to the NH₂ terminus containing the CAP-Gly motifs (Komarova et al., 2002a; Arnal et al., 2004). How CLIP-170 rescues a shrinking end is not clear. CLIP-170 can promote tubulin oligomerization (Diamantopoulos et al., 1999; Arnal et al., 2004), and it is likely that this property serves to increase the local concentration of tubulin substrate, thereby lowering the entropic barrier for the polymerization reaction. CLIP-170 in mammalian cells has only been found at growing plus ends, most likely as a result of copolymerization with tubulin subunits followed by its release from older segments (Diamantopoulos et al., 1999; Perez et al., 1999; Folker et al., 2005). When a microtubule end shrinks, CLIP-170 falls off. Is there a mechanism to get CLIP-170 close to the depolymerizing end and facilitate its function as a rescue factor? Given the proposed function of Stu2 as a rescue factor for spindle microtubules, one may easily imagine a similar scenario in which vesicle-bound CLIP-170 may be transported to the approaching microtubule end to rescue it from further shrinkage.If vesicle-bound CLIP-170 is transported to the plus end in a manner similar to Stu2, could such transport be mediated by plus end–directed kinesins? Although the kinesin involved in transporting Stu2 toward the microtubule plus end still needs to be identified, detailed image analyses have revealed a role for the Kip2/Tea2 kinesins (members of the kinesin-7 family) in transporting CLIP-170 homologues in fungi (Busch et al., 2004; Carvalho et al., 2004). Bik1 and Tip1 are the CLIP-170 homologues in budding and fission yeasts, respectively, and these proteins are found at microtubule plus ends, where they act as growth-promoting factors or anticatastrophe factors (Berlin et al., 1990; Brunner and Nurse 2000; Carvalho et al., 2004). In both yeasts, the Kip2/Tea2 kinesins bind to and comigrate with the CLIP-170 homologues along the microtubule toward the plus end (Busch et al., 2004; Carvalho et al., 2004). Kinesins have also been implicated in targeting other +TIPs to microtubule plus ends (Jimbo et al., 2002; Maekawa et al., 2003; Zhang et al., 2003; Wu et al., 2006). For example, the mammalian tumor suppressor protein APC (adenomatous polyposis coli) may be targeted to the plus end by KIF3A/KIF3B (a heterotrimeric kinesin II in the kinesin-2 family) as well as by other mechanisms (Jimbo et al., 2002; Nathke 2004; Slep et al., 2005). It will be interesting to see whether a similar transport process for CLIP-170 exists in higher eukaryotic cells. It is possible that such a mechanism would deliver just enough CLIP-170 to the shrinking plus end to initiate rescue. When microtubule growth is resumed, CLIP-170''s intrinsic higher affinity for tubulin subunits and lower affinity for the microtubule wall may allow these proteins to “treadmill” on the growing end (Perez et al., 1999; Folker et al., 2005).The regulation of CLIP-170 activity appears to be rather complex. CLIP-170 is most likely phosphorylated by multiple kinases, including FKBP12–rapamycin-associated protein (mTOR; Choi et al., 2002). Although phosphorylation by mTOR/FKBP 12–rapamycin-associated protein may stimulate CLIP-170''s microtubule binding, phosphorylation by other kinases may cause CLIP-170 to dissociate from microtubules (Rickard and Kreis 1996; Choi et al., 2002). In vivo, CLIP-170 has a closed conformation that is presumably inactive and an open conformation that may interact with microtubules and dynein regulators such as dynactin (Schroer 2004) and LIS1 (Morris et al., 1998; Lansbergen et al., 2004). It is possible that phosphorylation may regulate the conversion between these two forms, but the specific mechanism and the spatial regulation for this conversion have yet to be resolved. If CLIP-170 is indeed released from a membranous cargo to move to the plus end in order to serve as a rescue factor, it would be interesting to know when and/or where such a conformational switch occurs. Finally, other proteins may play redundant roles with CLIP-170 in vesicular trafficking, which may explain why a dramatic defect in vesicle/organelle distribution is not detected when the CLIP-170 level is lowered or when the gene is knocked out (Lansbergen et al., 2004; Akhmanova et al., 2005).+TIPs other than CLIP-170 may play a similar role in rescuing shrinking microtubule tracks. For example, the dynactin complex that links dynein to membranous cargoes and promotes the processive motion of dynein (Schroer 2004) may act as a rescue factor. The p150^Glued subunit of dynactin and CLIP-170 both contain CAP-Gly microtubule-binding motifs at their NH2 termini, although p150^Glued contains one, whereas CLIP-170 contains two such motifs. Dynactin has been shown to behave as a +TIP facilitating the capture of vesicular cargo for minus end–directed transport (Vaughan et al., 1999; 2002). The head domain of the p150^Glued subunit containing the CAP-Gly motif has been shown to promote rescue in vivo in the absence of endogenous CLIP-170, although the effect was much weaker than that caused by the exogenous CLIP-170 head domain (Kamarova et al., 2002a). In vitro studies showed that dynactin may promote nucleation during microtubule assembly (Ligon et al., 2003), which is consistent with it being a potential rescue factor. As shown with CLIP-170, this capacity to bring multiple tubulins together may help to overcome the entropic barrier of the polymerization reaction. Finally, cargo-bound dynactin may also use kinesin to get to the plus end. The p150^Glued subunit of dynactin has been shown to interact directly with the COOH terminus of KAP3, a subunit of the heterotrimeric kinesin II (a member of the kinesin-2 family) that also binds to APC (Jimbo et al., 2002; Deacon et al., 2003; Dell 2003). Although this binding is implicated in dynactin''s role as a cargo adaptor for kinesin II, it is possible, in theory, that a small amount of dynactin may use this connection to move to the plus end.The proposed hypothesis that +TIPs may be released from a membranous cargo to rescue a shrinking microtubule track may apply to both minus and plus end–directed transport. In addition, it is important to point out that this hypothesis does not exclude other mechanisms for rescuing long microtubule tracks. Rescue may occur stochastically, and, sometimes, +TIPs may participate in other ways such as mediating microtubule capture by the actin-rich cortex to stabilize the track (Wen et al., 2004; Galjart 2005). In some situations, microtubule dynamics are modulated by the direct binding of membranous cargo to the growing or shrinking plus ends of microtubules (Waterman-Storer and Salmon, 1998).Currently, the ability of CLIP-170 or other +TIPs to be released from a membranous cargo and to act as a rescue factor for a shrinking microtubule is just a hypothesis. Nevertheless, searching for proteins involved in the communication between a cargo and the approaching shrinking end of its microtubule track is clearly an endeavor worth pursuing.     

Dynactin polices two-way organelle traffic

How is the bidirectional motion of organelles controlled? In this issue, Deacon et al. (2003) reveal the unexpected finding that dynactin (previously known to control dynein-based motility) binds to kinesin II and regulates anterograde movement of Xenopus melanosomes. This result suggests that dynactin may be a key player in coordinating vesicle traffic in this system.The movement of intracellular cargo is essential for cell survival. In animal cells, membranous organelles are propelled through the cytoplasm by microtubule-based motor proteins. Anterograde movement toward microtubule plus ends at the cell periphery is driven by motor proteins of the kinesin superfamily, whereas retrograde movement toward minus ends at the cell center is largely accomplished by cytoplasmic dynein. In most cells, organelles do not travel smoothly in one direction but frequently switch between plus and minus end–directed travel. The net time spent traveling in the plus versus the minus end direction determines the steady-state distribution of an organelle population within a cell. A long-standing question for those studying organelle transport is how this bidirectional trafficking is coordinated. Is the binding of kinesin and dynein to vesicles mutually exclusive, or are these motors bound at the same time but with their activities coordinately regulated? What molecule(s) might be responsible for linking kinesin and dynein activities? In this issue, Vladimir Gelfand''s group (Deacon et al., 2003) addresses these questions by studying the motor proteins kinesin II and cytoplasmic dynein that move pigment granules in Xenopus melanophore cells. Their results are surprising; the dynactin complex, previously known to bind to cytoplasmic dynein and anchor it to organelles, also interacts with kinesin II and is necessary for plus end–directed motion. The ability of dynactin to physically interact with these two opposite polarity motors suggests that it may be the long sought-after molecular switch that coordinates bidirectional movement in this system.Previous studies hinted that the actions of dynein and kinesin may be controlled via dynactin. Dynactin is a large, multimeric protein complex. Its p150^Glued subunit has binding sites for both microtubules and the intermediate chain of dynein and is thought to be responsible for the association of dynein with many of its cargo organelles (Karki and Holzbaur, 1995; Vaughan and Vallee, 1995; Waterman-Storer et al., 1995). Curiously, the treatment of extruded squid axoplasm with antibodies against p150^Glued inhibited both the anterograde and retrograde movement of organelles along microtubules (Waterman-Storer et al., 1997). These antibodies were known to inhibit the interaction of dynactin with dynein, but their effect on anterograde movement was more difficult to explain. However, genetic studies yielded similar results. Martin et al. (1999) found that mutations in either p150^Glued, the cytoplasmic dynein heavy chain, or kinesin I inhibited both retrograde and anterograde fast axonal transport in Drosophila larvae. This phenotype potentially could be explained by stalled retrograde vesicles sterically blocking the movement of anterograde cargo, but the authors also suggested the possibility of a physical linkage between kinesin, dynein, and dynactin. This theory was further tested by tracking the movement of lipid droplets in Drosophila embryos (Gross et al., 2002b). A mild defect in the dynein heavy chain impaired several aspects of minus end–directed transport of lipid droplets: run lengths, velocities, and the opposing optical trap force required to halt droplet movement were all decreased. Surprisingly, this mutation produced similar effects on droplets moving toward the microtubule plus ends. Embryos expressing a mutant p150^Glued protein that partially impaired dynactin function also exhibited impaired movement in both the plus and minus end directions. Collectively, these results suggested that dynactin might be involved in coordinating the bidirectional movement of organelles. However, these studies did not provide a molecular explanation of how this mechanism might work.To study the mechanism of coordination of bidirectional vesicle movement, Deacon et al. (2003) used Xenopus melanophores due to the unique ability to experimentally control the directional movement of their pigmented melanosomes (Daniolos et al., 1990). Upon treatment of melanophores with melatonin, the cAMP concentration in the cytoplasm drops and the melanosomes move with a net minus end–directed bias and aggregate toward the cell center. Treatment with melanocyte-stimulating hormone (MSH)^* restores cAMP levels, and the melanosomes exhibit a plus end–directed bias and disperse throughout the cell. Aggregation is accomplished by cytoplasmic dynein (Nilsson and Wallin, 1997), whereas dispersion requires the combined actions of kinesin II and the actin-based motor myosin V (Rogers and Gelfand, 1998; Tuma et al., 1998; Gross et al., 2002a). Kinesin II is a heterotrimeric protein consisting of two motor subunits and a third nonmotor subunit known as kinesin-associated protein (KAP) (Cole et al., 1992). KAP is thought to be involved in binding kinesin II to its cargo, although the mechanism for this interaction is not known.The role of dynactin in melanosome transport was investigated by disrupting dynactin function via the overexpression of dynamitin (Echeverri et al., 1996), a crucial subunit that holds the dynactin complex together. To ensure that all observed melanosome movement occurred on the microtubule cytoskeleton, actin filaments were depolymerized with latrunculin B. Here, the authors report that melanosome movement to both the plus and minus ends of microtubules was inhibited by dynamitin overexpression, suggesting a role for dynactin in coordinating bidirectional movement. They considered whether this result might be explained if both kinesin II and dynein bound to dynactin and thereby docked onto membranes. To test this idea, kinesin II was immunoprecipitated with a series of antibodies, and the authors found that dynactin was pulled down along with this kinesin motor in all cases. The reverse experiment of immunoprecipitating with p150^Glued antibodies also brought down kinesin II. Blot overlays of purified melanosomes with p150^Glued detected an interaction with a 115-kD protein, the expected size of Xenopus KAP. Subsequent overlay and affinity pull-down experiments with purified proteins confirmed the direct binding of p150^Glued to KAP. By constructing a series of GST fusion proteins, Deacon et al. (2003) were able to map the site of this interaction to residues 600–811 of p150^Glued and the COOH-terminal domain of KAP. Interestingly, this region of p150^Glued also interacts with the dynein intermediate chain, which raised the question of whether kinesin II and dynein might compete for binding to dynactin. Using a blot overlay competition assay, the authors found that the COOH-terminal KAP domain blocked the binding of p150^Glued to the dynein intermediate chain, whereas the NH₂-terminal KAP domain, used as a control, did not. This result confirms that the two motors cannot bind dynactin simultaneously.If these biochemical results are relevant to melanosome movement, then overexpression of KAP should inhibit both anterograde and retrograde traffic. Indeed, overexpession of Xenopus KAP or just its COOH-terminal fragment inhibited bidirectional melanosome movement. As a control, NH₂-terminal KAP had no effect on retrograde movement and only a small effect on anterograde movement, perhaps due to interactions with the kinesin II motor subunits. Together, the results of Deacon et al. (2003) demonstrate that kinesin II, via its KAP subunit, binds to the p150^Glued subunit of dynactin and that this interaction is important for kinesin II–mediated movement of melanosomes.Although the authors identify the p150^Glued subunit of dynactin as a key player in coordinating the bidirectional movement of melanosomes, the mechanism is still unclear. Their biochemical results showing competitive binding to dynactin suggest that binding of kinesin II and dynein to melanosomes may be mutually exclusive events; however, previous work has shown that this is not the case. In a recent paper from the same authors (Gross et al., 2002a), as well as an earlier study from Reese and Haimo (Reese and Haimo, 2000), the relative amounts of kinesin II and dynein bound to purified melanosomes did not change when cells were treated with melatonin to stimulate aggregation or with MSH to stimulate dispersion. Thus, it is possible that proteins other than dynactin might bind kinesin II and dynein to melanosomes. This question also could be addressed by determining if motor binding to melanophores is diminished in cells overexpressing KAP or dynamitin. Unfortunately, Deacon et al. (2003) were not able to answer this question by biochemical isolation of melanosomes and motor quantitation because transfected cells were only a small percentage of the total population. Another possible model is that dynactin is not needed for recruiting kinesin II and dynein to melanosomes but is somehow involved in regulating the activation or organization of motors already bound to the membrane.Future studies will no doubt explore whether dynactin is involved in bidirectional transport in systems other than melanophores. In intraflagellar transport, kinesin II and cytoplasmic dynein 2 are involved in moving nonmembranous particles between the cell body and the tip of the flagella or cilia (Rosenbaum and Witman, 2002). It will be interesting to determine whether dynactin plays a role in this type of cargo transport. In neurons, kinesin I is responsible for moving organelles from the cell body to the axon terminal. As discussed above, Martin et al. (1999) found that mutations in either kinesin I heavy chain, dynein, or p150^Glued all produced the same phenotype in Drosophila larvae neurons, suggesting that dynactin may play a role in coordinating bidirectional movement in this system as well. Immunoprecipitation of neuronal p150^Glued, however, brought down only dynein but not kinesin I. This finding may result from the fact that kinesin I, which possesses a light chain unrelated to the KAP subunit, could be linked indirectly to dynactin by another protein. Thus, this study by Deacon et al. (2003) has opened up a new area of exploration and dynactin will undoubtedly receive closer scrutiny from kinesin researchers in the future.     

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.     

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.The sphingomyelin pathway is initiated by the hydrolysis of sphingomyelin to generate the second messenger ceramide.¹ Sphingomyelin hydrolysis is a major pathway for stress-induced ceramide generation. Neutral sphingomyelinase (nSMase) is activated by a variety of environmental stress conditions, such as heat shock,^{1, 2, 3} oxidative stress (hydrogen peroxide (H₂O₂), oxidized lipoproteins),¹ ultraviolet (UV) radiation,¹ chemotherapeutic agents,⁴ and β-amyloid peptides.^{5, 6} Cytokines, including tumor necrosis factor (TNF)-α,^{7, 8, 9} interleukin (IL)-1β,¹⁰ Fas ligand,¹¹ and their associated proteins, also trigger the activation of nSMase.¹² Membrane-bound Mg²⁺-dependent nSMase is considered to be a strong candidate for mediating the effects of stress and inflammatory cytokines on ceramide.³Among the four vertebrate nSMases, nSMase1 (SMPD2) was the first to be cloned and is localized in the endoplasmic reticulum (ER) and Golgi apparatus.¹³ Several studies have focused on the potential signaling roles of nSMase1, and some reports have suggested that nSMase1 is important for ceramide generation in response to stress.^{5, 6, 14, 15} In addition, nSMase1 is responsible for heat-induced apoptosis in zebrafish embryonic cultured (ZE) cells, and a loss-of-function study showed a reduction in ceramide generation, caspase-3 activation, and apoptosis in zebrafish embryos.¹⁶ However, nSMase1-knockout mice showed no lipid storage diseases or abnormalities in sphingomyelin metabolism.¹⁷ Therefore, the molecular mechanisms by which nSMase1 is activated have yet to be elucidated.Environmental stress and inflammatory cytokines^{1, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27} stimulate stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) signaling, which involves the sequential activation of members of the mitogen-activated protein kinase (MAPK) family, including MAPK/ERK kinase kinase (MEKK)1/MAPK kinase (MKK)4, and/or SAPK/ERK kinase (SEK)1/MKK7, JNK, and c-jun. Both the JNK and sphingomyelin signaling pathways coordinately mediate the induction of apoptosis.¹ However, possible crosstalk between the JNK and sphingomyelin signaling pathways has not yet been characterized. Previously, we used SDS-PAGE to determine that nSMase1 polypeptides migrated at higher molecular masses,¹⁶ suggesting that the sphingomyelin signaling pathway might cause the production of a chemically modified phosphorylated nSMase1, which is stimulated under stressed conditions in ZE cells.¹⁶ Here, we demonstrate that JNK signaling results in the phosphorylation of Ser-270 of nSMase1, which initiates ceramide generation and apoptosis. We also provide evidence for a signaling mechanism that integrates cytokine- and stress-activated apoptosis in vertebrate cells. We studied stress-induced ceramide generation in two cell types: ZE cells and human leukemia Jurkat T-lymphoid cells. Stress-induced apoptosis has been investigated in these systems previously.^{16, 28}     

Induction of COX-2-PGE2 synthesis by activation of the MAPK/ERK pathway contributes to neuronal death triggered by TDP-43-depleted microglia

Neuroinflammation is a striking hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Previous studies have shown the contribution of glial cells such as astrocytes in TDP-43-linked ALS. However, the role of microglia in TDP-43-mediated motor neuron degeneration remains poorly understood. In this study, we show that depletion of TDP-43 in microglia, but not in astrocytes, strikingly upregulates cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) production through the activation of MAPK/ERK signaling and initiates neurotoxicity. Moreover, we find that administration of celecoxib, a specific COX-2 inhibitor, greatly diminishes the neurotoxicity triggered by TDP-43-depleted microglia. Taken together, our results reveal a previously unrecognized non-cell-autonomous mechanism in TDP-43-mediated neurodegeneration, identifying COX-2-PGE2 as the molecular events of microglia- but not astrocyte-initiated neurotoxicity and identifying celecoxib as a novel potential therapy for TDP-43-linked ALS and possibly other types of ALS.Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the degeneration of motor neurons in the brain and spinal cord.¹ Most cases of ALS are sporadic, but 10% are familial. Familial ALS cases are associated with mutations in genes such as Cu/Zn superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TARDBP) and, most recently discovered, C9orf72. Currently, most available information obtained from ALS research is based on the study of SOD1, but new studies focusing on TARDBP and C9orf72 have come to the forefront of ALS research.^{1, 2} The discovery of the central role of the protein TDP-43, encoded by TARDBP, in ALS was a breakthrough in ALS research.^{3, 4, 5} Although pathogenic mutations of TDP-43 are genetically rare, abnormal TDP-43 function is thought to be associated with the majority of ALS cases.¹ TDP-43 was identified as a key component of the ubiquitin-positive inclusions in most ALS patients and also in other neurodegenerative diseases such as frontotemporal lobar degeneration,^{6, 7} Alzheimer''s disease (AD)^{8, 9} and Parkinson''s disease (PD).^{10, 11} TDP-43 is a multifunctional RNA binding protein, and loss-of-function of TDP-43 has been increasingly recognized as a key contributor in TDP-43-mediated pathogenesis.^{5, 12, 13, 14}Neuroinflammation, a striking and common hallmark involved in many neurodegenerative diseases, including ALS, is characterized by extensive activation of glial cells including microglia, astrocytes and oligodendrocytes.^{15, 16} Although numerous studies have focused on the intrinsic properties of motor neurons in ALS, a large amount of evidence showed that glial cells, such as astrocytes and microglia, could have critical roles in SOD1-mediated motor neuron degeneration and ALS progression,^{17, 18, 19, 20, 21, 22} indicating the importance of non-cell-autonomous toxicity in SOD1-mediated ALS pathogenesis.Very interestingly, a vital insight of neuroinflammation research in ALS was generated by the evidence that both the mRNA and protein levels of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) are upregulated in both transgenic mouse models and in human postmortem brain and spinal cord.^{23, 24, 25, 26, 27, 28, 29} The role of COX-2 neurotoxicity in ALS and other neurodegenerative disorders has been well explored.^{30, 31, 32} One of the key downstream products of COX-2, prostaglandin E2 (PGE2), can directly mediate COX-2 neurotoxicity both in vitro and in vivo.^{33, 34, 35, 36, 37} The levels of COX-2 expression and PGE2 production are controlled by multiple cell signaling pathways, including the mitogen-activated protein kinase (MAPK)/ERK pathway,^{38, 39, 40} and they have been found to be increased in neurodegenerative diseases including AD, PD and ALS.^{25, 28, 32, 41, 42, 43, 44, 45, 46} Importantly, COX-2 inhibitors such as celecoxib exhibited significant neuroprotective effects and prolonged survival or delayed disease onset in a SOD1-ALS transgenic mouse model through the downregulation of PGE2 release.²⁸Most recent studies have tried to elucidate the role of glial cells in neurotoxicity using TDP-43-ALS models, which are considered to be helpful for better understanding the disease mechanisms.^{47, 48, 49, 50, 51} Although the contribution of glial cells to TDP-43-mediated motor neuron degeneration is now well supported, this model does not fully suggest an astrocyte-based non-cell autonomous mechanism. For example, recent studies have shown that TDP-43-mutant astrocytes do not affect the survival of motor neurons,^{50, 51} indicating a previously unrecognized non-cell autonomous TDP-43 proteinopathy that associates with cell types other than astrocytes.Given that the role of glial cell types other than astrocytes in TDP-43-mediated neuroinflammation is still not fully understood, we aim to compare the contribution of microglia and astrocytes to neurotoxicity in a TDP-43 loss-of-function model. Here, we show that TDP-43 has a dominant role in promoting COX-2-PGE2 production through the MAPK/ERK pathway in primary cultured microglia, but not in primary cultured astrocytes. Our study suggests that overproduction of PGE2 in microglia is a novel molecular mechanism underlying neurotoxicity in TDP-43-linked ALS. Moreover, our data identify celecoxib as a new potential effective treatment of TDP-43-linked ALS and possibly other types of ALS.     

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.     

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.     

Synaptic dysfunction,memory deficits and hippocampal atrophy due to ablation of mitochondrial fission in adult forebrain neurons

Well-balanced mitochondrial fission and fusion processes are essential for nervous system development. Loss of function of the main mitochondrial fission mediator, dynamin-related protein 1 (Drp1), is lethal early during embryonic development or around birth, but the role of mitochondrial fission in adult neurons remains unclear. Here we show that inducible Drp1 ablation in neurons of the adult mouse forebrain results in progressive, neuronal subtype-specific alterations of mitochondrial morphology in the hippocampus that are marginally responsive to antioxidant treatment. Furthermore, DRP1 loss affects synaptic transmission and memory function. Although these changes culminate in hippocampal atrophy, they are not sufficient to cause neuronal cell death within 10 weeks of genetic Drp1 ablation. Collectively, our in vivo observations clarify the role of mitochondrial fission in neurons, demonstrating that Drp1 ablation in adult forebrain neurons compromises critical neuronal functions without causing overt neurodegeneration.In addition to their crucial importance in energy conversion, mitochondria serve many other housekeeping functions, including calcium buffering, amino-acid and steroid biosynthesis as well as fatty acids beta-oxidation and regulation of cell death. During the past decade, it has become increasingly clear that processes regulating mitochondrial morphology and ultrastructure are influenced by specific cellular requirements upon which mitochondria, in a precisely regulated manner, undergo fusion and division events.¹ Maintaining this balance is especially important for highly energy-consuming, polarized cells such as neurons, where single organellar units sprouting from the mitochondrial network are transported along the cytoskeleton into dendrites and spines to meet local energy requirements.² In addition, elaborate quality-control mechanisms also rely on mitochondrial dynamics: whereas defective organelles are sequestered by fission, enabling their removal from the mitochondrial network,^{3, 4} fusion supports qualitative homogeneity of the syncytium through complementation.⁵Mitochondrial fusion and fission are mediated by large GTPases of the dynamin superfamily.⁶ The outer mitochondrial membrane mitofusins 1 (MFN1) and 2 (MFN2) tether mitochondrial membranes by homodimer or heterodimer formation,⁷ thereby initiating fusion of the organelles, a process that also involves the inner mitochondrial membrane-associated GTPase Optic Atrophy 1.⁸ In addition, MFN2 also mediates contacts between mitochondria and endoplasmic reticulum.⁹ The only known mammalian mitochondrial fission protein, Dynamin-Related Protein 1 (Drp1), translocates upon dephosphorylation by calcineurin¹⁰ to fission sites where it binds to mitochondrial fission factor.¹¹ Drp1 translocation is preceded by ER membranes wrapping around mitochondria to constrict the organelles,¹² thereby facilitating the formation of multimeric Drp1 complexes that, upon GTP hydrolysis, further tighten to complete the process of mitochondrial fission.¹³Genetic evidence in mice and humans indicates that mitochondrial dynamics are crucially important in neurons: in humans, a sporadic dominant-negative DRP1 mutation caused a lethal syndromic defect with abnormal brain development;¹⁴ similarly, constitutive Drp1 knockout in the mouse brain leads to lethal neurodevelopmental defects.^{15, 16} Although the crucial role of Drp1 during brain development is undisputed, studies on Drp1 function in postmitotic (adult) neurons are scarce; likewise, Drp1 ablation studies in primary cultures have so far failed to yield a conclusive picture. In vitro, Drp1 ablation is reported to lead to a super-elongated neuroprotective^{17, 18, 19, 20, 21, 22, 23, 24} or an aggregated mitochondrial phenotype associated with neurodegeneration.^{15, 16, 25, 26, 27} These discrepancies are probably due to different experimental conditions: neuronal health is indeed influenced by the onset and duration of Drp1 inhibition, which varies considerably among the cited reports,²⁸ and different types of neuronal cultures studied display different sensitivity to Drp1 inhibition. In vivo, Drp1 ablation in Purkinje cells results in oxidative stress and neurodegeneration,²⁹ demonstrating that Drp1 is essential for postmitotic neurons'' health. In contrast, transient pharmacological Drp1 inhibition is neuroprotective in several mouse ischemia models, indicating that temporarily blocking mitochondrial fission holds therapeutic potential.^{30, 31, 32}To elucidate the consequences of blocked mitochondrial fission in the central nervous system in vivo, we bypassed the critical role of Drp1 during brain development by generating Drp1^flx/flx mice¹⁵ expressing tamoxifen-inducible Cre recombinase under the control of the CaMKIIα promoter.³³ Upon induced Drp1 deletion in postmitotic adult mouse forebrain neurons, mice develop progressive, neuronal subtype-specific alterations in mitochondrial shape and distribution in the absence of overt neurodegeneration. In addition, respiratory capacity, ATP content, synaptic reserve pool vesicle recruitment as well as spatial working memory are impaired, demonstrating that severely dysregulated mitochondrial dynamics can compromise critical neuronal functions in vivo without causing neuronal cell death.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

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.     

Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation

The deregulation of miR-101 and DNMT3a has been implicated in the pathogenesis of multiple tumor types, but whether and how miR-101 silencing and DNMT3a overexpression contribute to lung tumorigenesis remain elusive. Here we show that miR-101 downregulation associates with DNMT3a overexpression in lung cancer cell lines and patient tissues. Ectopic miR-101 expression remarkably abrogated the DNMT3a 3′-UTR luciferase activity corresponding to the miR-101 binding site and caused an attenuated expression of endogenous DNMT3a, which led to a reduction of global DNA methylation and the re-expression of tumor suppressor CDH1 via its promoter DNA hypomethylation. Functionally, restoration of miR-101 expression suppressed lung cancer cell clonability and migration, which recapitulated the DNMT3a knockdown effects. Interestingly, miR-101 synergized with decitabine to downregulate DNMT3a and to reduce DNA methylation. Importantly, ectopic miR-101 expression was sufficient to trigger in vivo lung tumor regression and the blockage of metastasis. Consistent with these phenotypes, examination of xenograft tumors disclosed an increase of miR-101, a decrease of DNMT3a and the subsequent DNA demethylation. These findings support that the loss or suppression of miR-101 function accelerates lung tumorigenesis through DNMT3a-dependent DNA methylation, and suggest that miR-101-DNMT3a axis may have therapeutic value in treating refractory lung cancer.Owing to a high propensity for recurrence and a high rate of metastasis at the advanced stages,^{1, 2, 3} lung cancer remains the leading cause of cancer-related mortality. DNA methylation is a major epigenetic rule controlling chromosomal stability and gene expression.^{4, 5} It is under control of DNA methyltransferases (DNMTs), whose overexpression in lung cancer cells predicts worse outcomes.^{6, 7} It is postulated that DNMT overexpression induces DNA hypermethylation and silencing of tumor suppressor genes (TSGs), leading to an aggressive lung cancer. Indeed, enforced expression of DNMT1 or DNMT3a increases DNA methylation, while the abolition of DNMT expression by genetic depletion, microRNAs (miRs) or small molecules reduces genome-wide and gene-specific DNA methylation and restores TSG expression.^{8, 9, 10, 11, 12, 13} As TSGs are the master controllers for cell multiplicity and their silencing predicts poor prognosis,^{14, 15} TSG re-expression via promoter DNA hypomethylation inhibits cell proliferation and induces cell differentiation.^{13, 16} Thus, DNMT gene abundance could serve as a target for anticancer therapy, but how DNMT upregulation occurs in lung cancer is incompletely understood.MiRs are small non-coding RNAs that crucially regulate target gene expression. Up to 30% of all protein-coding genes are predicted to be targeted by miRs,^{17, 18} supporting the key roles of miRs in controlling cell fate.^{19, 20, 21, 22} Research is showing that certain miRs are frequently dysregulated in cancers, including lung cancer.^{7, 23, 24} As miR targets can promote or inhibit cancer cell expansion, miRs have huge potential for acting as bona fide oncogenes (i.e., miR-21) or TSGs (i.e., miR-29b).^{7, 25} We and others demonstrated that the levels of DNMT1 or DNMT3a or DNMT3b are regulated by miR-29b, miR-148, miR-152 or miR-30c,^{7, 13, 26, 27} and overexpression of these miRs results in DNA hypomethylation and TSG reactivation with the concurrent blockage of cancer cell proliferation.^{7, 13} These findings underscore the importance of miRs as epigenetic modulators and highlight their therapeutic applications.MiR-101 is frequently silenced in human cancers^{28, 29, 30, 31} and, importantly, exhibits antitumorigenic properties when overexpressed. Mechanistically, miR-101 inactivation by genomic loss causes the overexpression of EZH2, a histone methyltransferase, via 3′-UTR targeting, which is followed by histone hypermethylation and aggressive tumorigenesis.^{29, 30, 32} However, whether and how miR-101 silencing contributes to DNA hypermethylation patterning in lung cancer is unclear. In this study, we explore the role of miR-101 in regulating DNMT3a expression and the impacts of miR-101-DNMT3a nexus on lung cancer pathogenesis. We showed that the expression of miR-101 and DNMT3a was negatively correlated in lung cancer. We presented evidence that ectopic miR-101 expression decreased DNMT3a levels, reduced global DNA methylation and upregulated CDH1 via its promoter DNA demethylation. The biological significance of miR-101-mediated DNA hypomethylation and CDH1 re-expression was evident by its inhibition of lung tumor cell growth in vitro and in vivo. Thus, our findings mechanistically and functionally link miR-101 silencing to DNA hypermethylation in lung cancer cells.     

Safety and Colonization of Two Novel virG(icsA)-based Live Shigella sonnei Vaccine Strains in Rhesus Macaques (Macaca mulatta)

Shigella are gram-negative bacterium that cause bacillary dysentery (shigellosis). Symptoms include diarrhea and discharge of bloody mucoid stools, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Persons traveling to regions with poor sanitation and crowded conditions become particularly susceptible to shigellosis. Currently a vaccine for Shigella has not been licensed in the United States, and the organism quickly becomes resistant to medications. During the past 10 y, several live attenuated oral Shigella vaccines, including the strain WRSS1, have been tested in humans with considerable success. These Phase I vaccines lack the gene for the protein VirG also known as IcsA, which enables the organism to disseminate in the host target tissue. However, 5% to 20% of the vaccinated volunteers developed mild fever and brief diarrhea, and the removal of additional virulence-associated genes from the vaccine strain may reduce or eliminate these side effects. We administered 2 Shigella sonnei vaccines, WRSs2 and WRSs3, along with WRSS1 to compare their rates of colonization and clinical safety in groups of 5 rhesus macaques. The primate model provides the most physiologically relevant animal system to test the validity and efficacy of vaccine candidates. In this pilot study using a gastrointestinal model of infection, the vaccine candidates WRSs2 and WRSs3, which have additional deletions in the enterotoxin and LPS modification genes, provided better safety and comparable immunogenicity to those of WRSS1.Abbreviations: IcsA, intercellular spread protein A (VirG protein); Ipa, invasion plasmid antigen; LPS, lipopolysaccharideVarious species of Shigella cause bacillary dysentery, also known as shigellosis, in humans and other primates.^{1,18,31,37,49} Symptoms include diarrhea, with various degrees of mucus and hematochezia, accompanied by severe abdominal pain, nausea, vomiting, malaise, and fever. Shigella is a gram-negative bacterium whose genomic sequence is very similar to that of Escherichia coli and is phylogenetically considered a pathotype of E. coli.²³ Shigella infections are spread by the fecal–oral route through the consumption of contaminated food and water or by mechanical vectors such as insects. Persons traveling in areas with poor sanitation and crowded conditions become particularly susceptible to such diseases.^2,39,48,56 In colonies of research macaques, Shigella infections are spread by the fecal–oral route, originating from addition of animals that are asymptomatic carriers, either from the wild or from other colonies.^1,55 Currently no Shigella vaccine has been licensed in the United States, although several are under development.^{25,32,35,38,54} Antibiotics are used as treatment therapy, but many Shigella isolates are multidrug-resistant, increasing the need for a preventive vaccine for travelers, military personnel, and children in endemic areas.^{16,34,41,44,57} Rhesus macaques represent an excellent model for studying Shigella because primates are the only known animal model that simulate natural human infection, including dysentery after oral challenge.^{11-13,17,28,40,47,46} Other animal models, such as the mouse pulmonary model and the guineapig ocular and intranasal models, mimic specific, isolated steps in Shigella pathogenesis.^14,19,26There are 4 major serogroups of Shigella and one or more serotypes within each serogroup. This classification is based on antigenic differences in the O-antigen polysaccharide component of the outer membrane-associated lipopolysaccharide (LPS). Estimates from the Centers for Disease Control (Atlanta, GA) indicate that approximately 400,000 cases of shigellosis occur in the US annually, with an estimated 165 million cases worldwide each year.²² These occur predominantly in developing countries, where the population most affected is children younger than 5 y.²²Various in vitro cell culture models of infection, as well as studies in animal models including gastrointestinal infection in nonhuman primates, have contributed to the current understanding of Shigella pathogenesis.^4,42 Shigella organisms target the distal region of the colon and rectum, where the bacteria are captured by specialized M-cells located within the follicle-associated epithelium. The M-cells deliver bacterial antigens, which include bacterial LPS and invasion plasmid antigen (Ipa) proteins, to the underlying antigen-presenting macrophages and dendritic cells.³⁶ Virulent Shigella strains escape macrophages by a cytotoxic effect, causing release of the bacteria and inflammatory cytokines such as IL1β, TNFα, and IL18. The bacteria then invade adjacent intestinal epithelial cells at the basolateral side, through interaction between bacterial proteins and multiple signaling molecules within the host cell.^36,42Shigella are capable of orchestrating this uptake into nonphagocytic epithelial cells, a process termed invasion, through the secretion of proteins that are highly conserved among all virulent strains. These proteins are encoded by a large, 213-kb plasmid, referred to as the invasion plasmid or the virulence plasmid.^50-52 Each bacterium ultimately is engulfed within an endocytic vacuole. Subsequent lysis of the vacuole sets the bacterium free within the epithelial cell cytoplasm, where it replicates and moves to adjacent epithelial cells, with the help of the intercellular spread (Ics) A protein, also known as VirG.^3,24,45 The VirG(IcsA)-assisted intra- and intercellular spread of the bacteria within the epithelial tissue contributes significantly to the loss of epithelial cell integrity and accompanying tissue injury. Shigella strains with loss of the virG(icsA) gene are significantly attenuated in all animal models of virulence.⁵ Several Shigella vaccine candidates that have undergone Phase 1 clinical trials are principally attenuated due to lack of the VirG(IcsA) protein, these include S. flexneri 2a strain SC602, S. sonnei strain WRSS1, and S. dysenteriae 1 vaccine strain WRSd1.^{5,10,15,21,53}The S. sonnei first-generation vaccine candidate WRSS1 has been tested in several Phase I inpatient and outpatient trials.^15,21,33 WRSS1, like the previous S. flexneri 2a vaccine candidate, SC602, was shown to be safe in Phase I trials when orally administered at a dose of 10³ to 10⁴ CFU.^15,21,33 Both SC602 and WRSS1 colonized well as evidenced by fecal excretion of the vaccine strain for 5 to 7 d. The vigorous immune response seen with both these vaccine candidates was correlated with the robust colonization. However, 5% to 20% of the SC602- and WRSS1-immunized volunteers showed mild and transient symptoms of diarrhea and fever. In light of new knowledge about Shigella pathogenesis as well as data from other clinical trials, 2 new and presumably safer S. sonnei derivatives have been constructed (WRSs2 and WRSs3). In addition to loss of virG(icsA), WRSs2 has deletions in 2 genes, senA and senB, that are present on the virulence plasmid.^29,52 senA (also known as shet2-1 and ospD3) encodes the enterotoxin ShET2-1, which has been shown to cause fluid accumulation in rabbit ileal loops.^7,8 senB (also known as shet2-2 and ospD2) encodes a similarly sized protein as SenA and shows 40% identity at the amino acid level. The enterotoxic activity of ShET2-2 remains to be demonstrated.^20,29 Like WRSs2, WRSs3 lacks virG(icsA) and the 2 enterotoxin genes with the additional deletion of the virulence plasmid-based msbB2 gene. Lack of the msbB2 gene product has previously been shown to produce a less-toxic LPS molecule, which might help to reduce the symptoms of fever seen in WRSS1-immunized volunteers.^20,29 How these additional mutations, especially that of msbB2, will affect colonization of these strains is unclear, given that effective colonization is critical in the elicitation of immune response and protection. The primary goal of this pilot study was to compare the safety and colonization of 2 novel S. sonnei vaccine candidate strains (WRSs2 and WRSs3), with those of the clinically tested vaccine strain WRSS1, by using a gastrointestinal model of infection. The hypothesis tested was that the 2 novel Shigella vaccine strains would colonize the vaccinated animal and induce an immune response similar to that of WRSS1 but with less frequent and less severe clinical side effects.