FAIM-L - SIVA-1: Two Modulators of XIAP in Non-Apoptotic Caspase Function

Apoptosis is crucial for the correct development of the nervous system. In adulthood, the same protein machinery involved in programmed cell death can control neuronal adaptiveness through modulation of synaptic pruning and synaptic plasticity processes. Caspases are the main executioners in these molecular pathways, and their strict regulation is essential to perform neuronal remodeling preserving cell survival. FAIM-L and SIVA-1 are regulators of caspase activation. In this review we will focus on FAIM-L and SIVA-1 as two functional antagonists that modulate non-apoptotic caspase activity in neurons. Their participation in long-term depression and neurite pruning will be described in base of the latest studies performed. In addition, the association of FAIM-L non-apoptotic functions with the neurodegeneration process will be reviewed.

Infection of Galleria mellonella (Lepidoptera) Larvae With the Entomopathogenic Fungus Conidiobolus coronatus (Entomophthorales) Induces Apoptosis of Hemocytes and Affects the Concentration of Eicosanoids in the Hemolymph

Apoptosis and autophagy, the mechanisms of programmed cell death, play critical roles in physiological and pathological processes in both vertebrates and invertebrates. Apoptosis is also known to play an important role in the immune response, particularly in the context of entomopathogenic infection. Of the factors influencing the apoptotic process during infection, two of the lesser known groups are caspases and eicosanoids. The aim of this study was to determine whether infection by the entomopathogenic soil fungus Conidiobolus coronatus is associated with apoptosis and changes in caspase activity in the hemocytes of Galleria mellonella larvae, and to confirm whether fungal infection may affect eicosanoid levels in the host. Larvae were exposed for 24 h to fully grown and sporulating fungus. Hemolymph was collected either immediately after termination of exposure (F24 group) or 24 h later (F48 group). Apoptosis/necrosis tests were performed in hemocytes using fluorescence microscopy and flow cytometry, while ELISA tests were used to measure eicosanoid levels. Apoptosis and necrosis occurred to the same degree in F24, but necrosis predominated in F48. Fungal infection resulted in caspase activation, increased PGE1, PGE2, PGA1, PGF2α, and 8-iso-PGF2α levels and decreased TXB2 levels, but had no effect on TXA2 or 11-dehydro-TXB2 concentrations. In addition, infected larvae demonstrated significantly increased PLA2 activity, known to be involved in eicosanoid biosynthesis. Our findings indicate that fungal infection simultaneously induces apoptosis in insects and stimulates general caspase activity, and this may be correlated with changes in the concentrations of eicosanoids.

The Cellular and Physiological Basis of Host-Symbiont Specificity in a Model Cnidarian-Dinoflagellate Symbiosis

<p>The ability of corals to form novel partnerships with symbionts that may be better suited to new environmental conditions is an important factor when assessing the ability for corals to adapt to climate change. However, relatively little attention has been given to the effects of hosting different symbiont types on holobiont physiology, competitive interactions between these symbionts, or the capacity of the host to regulate populations of different symbionts. Such factors likely play an important role in patterns of host-symbiont specificity and flexibility, and hence the potential for corals to respond to climate change. The aim of this research was to characterise the cellular and physiological events associated with hosting different symbiont species in Exaiptasia pallida (commonly referred to as ‘Aiptasia’), a model cnidarian-dinoflagellate symbiosis, and how these events might contribute to host-symbiont specificity. The specific objectives were to: (1) determine the effect of symbiont species on the population dynamics of host colonisation and holobiont physiology; (2) measure the competitiveness of the homologous symbiont versus heterologous symbionts, under both control and elevated temperatures; and quantify the ability of the host to regulate its symbiont population in response to homologous versus heterologous symbiont taxa (3) via host-cell apoptosis and (4) via symbiont cell cycle regulation. To explore this, aposymbiotic (i.e. symbiont-free) individuals of Aiptasia were first inoculated with one of five Symbiodinium taxa (the homologous S. minutum or heterologous S. microadriaticum, phylotype C3, S. trenchii or S. voratum), and the rates and patterns of colonisation assessed. Proliferation success inside the anemone was different between symbionts, with the homologous S. minutum being the most successful species, while Symbiodinium C3 and S. voratum struggled or failed to form a long-lasting symbiosis. The spatial pattern of symbiont colonisation was identical for all the successful Symbiodinium taxa, however the timing differed between these different symbionts. Symbiont identity also had an effect on holobiont fitness, as S. microadriaticum and S. trenchii were less beneficial to the host compared to S. minutum, as indicated by lower rates of photosynthesis, anemone growth and pedal laceration (i.e. asexual reproduction). The taxon-specific differences demonstrated here provided a basis for the subsequent thesis chapters, leading to questions about how the different symbionts might compete with one another and be regulated by the host. The competitiveness of the homologous symbiont relative to heterologous ones, and hence the ability of the host to ‘switch’ and ‘shuffle’ its symbiont population, was tested by inoculating aposymbiotic sea anemones either with simultaneous or sequential mixtures of thermally tolerant and sensitive Symbiodinium and exposing them to control versus elevated temperatures. The homologous species was dominant regardless of temperature, outcompeting the heterologous, thermally tolerant S. microadriaticum and S. trenchii. This result indicates that the high level of specificity seen between Aiptasia and S. minutum in the Pacific Ocean may result, in part, from a reluctance to form new symbioses, even if such associations have the potential to confer a degree of thermal tolerance that may be beneficial under future climate change. The differential success of the different symbionts was also reflected in the host’s apoptotic response to their presence in its tissues, as measured via caspase-3 activity. In particular, anemones hosting S. minutum and S. microadriaticum exhibited lower levels of caspase activity than those hosting S. trenchii and S. voratum throughout symbiosis establishment, consistent with symbiont proliferation success. The general pattern of caspase activity during the 28-days colonisation period was similar, however, with induction of caspase-3 activity upon inoculation, followed by a marked decline in activity over the subsequent week, and then an increase (either moderate or marked depending upon symbiont identity) across the remainder of the colonisation period measured. Host cell apoptosis therefore likely plays an important role in determining the compatibility and fate of different Symbiodinium taxa in a host, and the potential for establishing novel symbioses. In contrast to the apparent importance of host apoptosis, symbiont cell cycle control did not seem to play an important role in determining the different rates of symbiont colonisation observed. Flow cytometry was used to determine the relative proportion of cells in the different phases of the cell cycle (i.e. G1, G2, S, M), with all symbiont taxa exhibiting the same pattern of cell cycle progression. In particular, more cells were in the S and G2/M phases combined than in G1 during the first two weeks of colonisation, but this changed as colonisation progressed, when a greater proportion cells were in the G1 phase. This indicates that symbiont cell division becomes limited in the later stages of colonisation as symbiont density increases, consistent with increasing resource limitation. This thesis provides valuable insights into the regulation of the cnidarian-dinoflagellate symbiosis, and the events that contribute to host-symbiont specificity. In particular, it suggests that through cellular control and physiological impacts on the host (and hence the overall symbiosis), there is likely limited potential to establish new host-symbiont partnerships that allow for adaptation to our warming climate. The next step is now to further elucidate the relative importance of post-phagocytosis control mechanisms, and to test the generality of my findings by extending them from the model Aiptasia system to reef corals.</p>

The Cellular and Physiological Basis of Host-Symbiont Specificity in a Model Cnidarian-Dinoflagellate Symbiosis

<p>The ability of corals to form novel partnerships with symbionts that may be better suited to new environmental conditions is an important factor when assessing the ability for corals to adapt to climate change. However, relatively little attention has been given to the effects of hosting different symbiont types on holobiont physiology, competitive interactions between these symbionts, or the capacity of the host to regulate populations of different symbionts. Such factors likely play an important role in patterns of host-symbiont specificity and flexibility, and hence the potential for corals to respond to climate change. The aim of this research was to characterise the cellular and physiological events associated with hosting different symbiont species in Exaiptasia pallida (commonly referred to as ‘Aiptasia’), a model cnidarian-dinoflagellate symbiosis, and how these events might contribute to host-symbiont specificity. The specific objectives were to: (1) determine the effect of symbiont species on the population dynamics of host colonisation and holobiont physiology; (2) measure the competitiveness of the homologous symbiont versus heterologous symbionts, under both control and elevated temperatures; and quantify the ability of the host to regulate its symbiont population in response to homologous versus heterologous symbiont taxa (3) via host-cell apoptosis and (4) via symbiont cell cycle regulation. To explore this, aposymbiotic (i.e. symbiont-free) individuals of Aiptasia were first inoculated with one of five Symbiodinium taxa (the homologous S. minutum or heterologous S. microadriaticum, phylotype C3, S. trenchii or S. voratum), and the rates and patterns of colonisation assessed. Proliferation success inside the anemone was different between symbionts, with the homologous S. minutum being the most successful species, while Symbiodinium C3 and S. voratum struggled or failed to form a long-lasting symbiosis. The spatial pattern of symbiont colonisation was identical for all the successful Symbiodinium taxa, however the timing differed between these different symbionts. Symbiont identity also had an effect on holobiont fitness, as S. microadriaticum and S. trenchii were less beneficial to the host compared to S. minutum, as indicated by lower rates of photosynthesis, anemone growth and pedal laceration (i.e. asexual reproduction). The taxon-specific differences demonstrated here provided a basis for the subsequent thesis chapters, leading to questions about how the different symbionts might compete with one another and be regulated by the host. The competitiveness of the homologous symbiont relative to heterologous ones, and hence the ability of the host to ‘switch’ and ‘shuffle’ its symbiont population, was tested by inoculating aposymbiotic sea anemones either with simultaneous or sequential mixtures of thermally tolerant and sensitive Symbiodinium and exposing them to control versus elevated temperatures. The homologous species was dominant regardless of temperature, outcompeting the heterologous, thermally tolerant S. microadriaticum and S. trenchii. This result indicates that the high level of specificity seen between Aiptasia and S. minutum in the Pacific Ocean may result, in part, from a reluctance to form new symbioses, even if such associations have the potential to confer a degree of thermal tolerance that may be beneficial under future climate change. The differential success of the different symbionts was also reflected in the host’s apoptotic response to their presence in its tissues, as measured via caspase-3 activity. In particular, anemones hosting S. minutum and S. microadriaticum exhibited lower levels of caspase activity than those hosting S. trenchii and S. voratum throughout symbiosis establishment, consistent with symbiont proliferation success. The general pattern of caspase activity during the 28-days colonisation period was similar, however, with induction of caspase-3 activity upon inoculation, followed by a marked decline in activity over the subsequent week, and then an increase (either moderate or marked depending upon symbiont identity) across the remainder of the colonisation period measured. Host cell apoptosis therefore likely plays an important role in determining the compatibility and fate of different Symbiodinium taxa in a host, and the potential for establishing novel symbioses. In contrast to the apparent importance of host apoptosis, symbiont cell cycle control did not seem to play an important role in determining the different rates of symbiont colonisation observed. Flow cytometry was used to determine the relative proportion of cells in the different phases of the cell cycle (i.e. G1, G2, S, M), with all symbiont taxa exhibiting the same pattern of cell cycle progression. In particular, more cells were in the S and G2/M phases combined than in G1 during the first two weeks of colonisation, but this changed as colonisation progressed, when a greater proportion cells were in the G1 phase. This indicates that symbiont cell division becomes limited in the later stages of colonisation as symbiont density increases, consistent with increasing resource limitation. This thesis provides valuable insights into the regulation of the cnidarian-dinoflagellate symbiosis, and the events that contribute to host-symbiont specificity. In particular, it suggests that through cellular control and physiological impacts on the host (and hence the overall symbiosis), there is likely limited potential to establish new host-symbiont partnerships that allow for adaptation to our warming climate. The next step is now to further elucidate the relative importance of post-phagocytosis control mechanisms, and to test the generality of my findings by extending them from the model Aiptasia system to reef corals.</p>

Alpha-Ketoglutarate and 5-HMF: A Potential Anti-Tumoral Combination against Leukemia Cells

We have recently shown that a combined solution containing alpha-ketoglutarate (aKG) and 5-hydroxymethyl-furfural (5-HMF) might have anti-tumoral potential due to its antioxidative activities. The question arises if these substances have caspase-3- and apoptosis-activating effects on the cell proliferation in Jurkat and HF-SAR cells. Antioxidative capacity of several combined aKG + 5-HMF solution was estimated by cigarette smoke radical oxidized proteins of fetal calf serum (FCS) using the estimation of carbonylated proteins. The usage of 500 µg/mL aKG + 166.7 µg/mL 5-HMF showed the best antioxidative capacity to inhibit protein modification of more than 50% compared to control measurement. A Jurkat cell line and human fibroblasts (HF-SAR) were cultivated in the absence or presence of combined AKG + 5-HMF solutions between 0 µg/mL aKG + 0 µg/mL 5-HMF and different concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF. Aliquots of Jurkat cells were tested for cell proliferation, mitochondrial activity, caspase activity, apoptotic cells and of the carbonylated protein content as marker of oxidized proteins in cell lysates after 24, 48, and 72 h of incubation. The combined solutions of aKG + 5-HMF were shown to cause a reduction in Jurkat cell growth that was dependent on the dose and incubation time, with the greatest reductions using 500 µg/mL aKG + 166.7 µg/mL 5-HMF after 24 h of incubation compared to 24 h with the control (22,832 cells vs. 32,537 cells), as well as after 48 h (21,243 vs. 52,123 cells) and after 72 h (23,224 cells). Cell growth was totally inhibited by the 500 µg/mL AKG + 166.7 µg/mL solution between 0 and 72 h of incubation compared to 0 h of incubation for the control. The mitochondrial activity measurements supported the data on cell growth in Jurkat cells: The highest concentration of 500 µg/mL aKG + 166.7 µg/mL 5-HMF was able to reduce the mitochondrial activity over 24 h (58.9%), 48 h (28.7%), and 72 h (9.9%) of incubation with Jurkat cells compared not only to the control incubation, but also to the concentrations of 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 375 µg/mL aKG 125 µg/mL 5-HMF, which were able to significantly reduce the mitochondrial activity after 48 h (28.7% or 35.1%) and 72 h (9.9% or 18.2%) compared to 24 h with the control (100%). A slight increase in cell proliferation was found in HF-SAR using the highest concentration (500 µg/mL aKG + 166.7 µg/mL 5-HMF) between 0 h and 72 h incubation of 140%, while no significant differences were found in the mitochondrial activity of HF-SAR in the absence or presence of several combined aKG + 5-HMF solutions. The solutions with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF showed a significantly higher caspase activity (51.6% or 13.5%) compared to the control (2.9%) in addition to a higher apoptosis rate (63.2% or 31.4% vs. control: 14.9%). Cell lysate carbonylated proteins were significantly higher in Jurkat cells compared to HF-SAR cells (11.10 vs. 2.2 nmol/mg). About 72 h incubation of Jurkat cells with 500 µg/mL aKG + 166.7 µg/mL 5-HMF or 250 µg/mL aKG + 83.3 µg/mL 5-HMF reduced significantly the carbonylated protein content down to 5.55 or 7.44 nmol/mg whereas only the 500 µg/mL aKG + 166.7 µg/mL 5-HMF solution showed a significant reduction of carbonylated proteins of HF-SAR (1.73 nmol/mg).

New Anti-Cancer Drug Compounds to Treat Mutated Waldenstrom Myd88 L265P

Waldenstrom Macroglobulinemia (WM) is a non-Hodgkin lymphoma, often associated with production of monoclonal IgM in a large amount. The increased level of IgM leads to the increased level of blood viscosity, potentially causing spontaneous bleeding, headaches, vertigo and could lead to stroke and coma. WM is a rare disease, affecting around 3 cases per million per year in the USA. Different chromosomal abnormalities can be cause of WM, but the most common mutation detected in WM is the mutation L265P on the Myd88 protein, a downstream regulator of TLR4 pathway. While today there are many treatment options to manage WM, including plasmapheresis, monoclonal (rituximab) and immunomodulating drugs (halidomide), cytokines, mTOR inhibitors (everolimus), Burton tyrosine kinase (Ibrutinib), there isn't a single effective enough treatment for WM that are widely used. In this study, MWCL-1 cells (adult WM), expressing the Myd88 L265P mutation, were used to test different drugs to monitor their effect in cell proliferation and apoptosis activities, in comparison to midostaurin (an inhibitor of FLT3), for 24 hours treatment. MWCL-1 cells were cultured in RPMI1640, in presence of 2, 10 and 25 % of fetal bovine serum (FBS). 25 % FBS was tested to be as close as possible with the level of serum present in human blood. Adipose stromal cells (ASC) were used as a reference control, in absence of serum. Compounds A (targeting TLR pathway, through IRAK-4) was used at various concentrations (0.05 to 100 µM), over 24h of treatment, in combination or not with compound B (targeting the DNA). Compound A has a high affinity for IRAK4, downstream kinase of TLR pathway. TLR pathway activation in leukemia, through Myd88 and IRAK4, is involved in the pro-inflammation response but it is also inducing proliferation and cell survival. Compound B is an inhibitor of the poly (ADP-ribose) polymerase (PARP), which are enzymes that are involved in DNA transcription, cell cycle regulation and DNA repair. In the ASC, the caspase activity of compound A was not different from the control, but the number of cells was lower at 50 and 100 µM, indicating that the compound A was delaying the cell growth. The number of MWCL-1 cells decreased only at higher compound A concentration (50 and 100 µM), during a 24h treatment, at 2, 10 and 25 % FBS. When the cells were treated with the compound A, the level of caspase 3/7 activity was elevated only at the highest concentration (50 and 100 µM), which is consistent with the decreased number of cells, at 2, 10 and 25 % FBS. MWCL-1 were more resistant to the anti-cancer effect of the compound A at 25 % FBS, indicating that it could work on patients because 25 % FBS is close to the patient's serum level. Midostaurin, from 0.05 to 100 µM, was reducing the number of cells and increasing the caspase 3/7 activity in the cells, in a dose response manner, at 2, 10 and 25 % FBS. However, data suggest that at the highest midostaurin dose (100 µM), compound A was more efficient than midostaurin (100 µM). Compound B alone, at 10 µM, had no effect on the MWCL-1 cell number and on the caspase 3/7 activity, for the 2, 10 and 25 % FBS. However, a synergistic effect was demonstrated when compound B (10 µM) was combined with compound A at 50 and 100 µM. The combination decreased the number of MWCL-1 cells and increased the caspase activity 3/7, more so than it was compared to the single compound studies. Our data suggest that it would be possible to lower the posology of the compound A when it will be given to the patients, in combination with the compound B. In conclusion, the data showed compound A alone or combined with the compound B decreased the level of proliferation and increased the level of apoptosis of MWCL-1 cells, cultured with 2 to 25 % of FBS. The use of compound A at high concentration to reduce the number of MWCL-1 reflect the difficulties to treat WM patient with the existing approved drugs. Further studies will be necessary to understand more the molecular mechanism affected by the compound A and/or B in the TLR-IRAK4 pathway, and to study their effect in vivo. Disclosures Oliva: Emmaus Lifesciences, Inc.: Current Employment. Villanueva: Emmaus Lifesciences, Inc.: Current Employment. Ochiai: Emmaus Lifesciences, Inc.: Ended employment in the past 24 months. Niihara: Emmaus Lifesciences, Inc.: Current Employment.

Comparative transcriptomic analysis reveals gene regulation mediated by caspase activity in a chordate organism

Background Apoptosis is a caspase regulated cell death present in all metazoans defined by a conserved set of morphological features. A well-described function of apoptosis is the removal of excessive cells during development and homeostasis. Recent studies have shown an unexpected signalling property of apoptotic cells, affecting cell fate and/or behaviour of neighbouring cells. In contrast to the apoptotic function of cell elimination, this new role of apoptosis is not well understood but seems caspase-dependent. To deepen our understanding of apoptotic functions, it is necessary to work on a biological model with a predictable apoptosis pattern affecting cell fate and/or behaviour. The tunicate Ciona intestinalis has a bi-phasic life cycle with swimming larvae which undergo metamorphosis after settlement. Previously, we have shown that the tail regression step during metamorphosis, characterized by a predictable polarized apoptotic wave, ensures elimination of most tail cells and controls primordial germ cells survival and migration. Results We performed differential transcriptomic analysis between control metamorphosing larvae and larvae treated with the pan-caspase inhibitor Z-VAD-fmk in order to explore the transcriptional control of apoptotic cells on neighbouring cells that survive and migrate. When caspase activity was impaired, genes known to be involved in metamorphosis were downregulated along with other implicated in cell migration and survival molecular pathways. Conclusion We propose these results as a confirmation that apoptotic cells can control surrounding cells fate and as a reference database to explore novel apoptotic functions in animals, including those related to migration and differentiation.

Effects of common Gram-negative pathogens causing male genitourinary-tract infections on human sperm functions

Male genitourinary tract (MGT) bacterial infections are considered responsible for 15% of male infertility, but the mechanisms underlying decreased semen quality are poorly known. We evaluated in vitro the effect of strains of Gram-negative uropathogenic species (two E.coli strains, three K. pneumoniae strains, P. aeruginosa and E. cloacae) on motility, viability, mitochondrial oxidative status, DNA fragmentation and caspase activity of human spermatozoa. All strains, except P. aeruginosa, reduced significantly sperm motility, with variable effects. Sperm Immobilizing Factor (SIF) was largely responsible for deteriorating effects on sperm motility of E. coli strains since they were completely reverted by knockout of SIF coding recX gene. Sequence alignment for RecX showed the presence of high homologous sequences in K. pneumoniae and E. cloacae but not in P. aeruginosa. These results suggest that, in addition to E.coli, other common uropathogenic Gram-negative bacteria affect sperm motility through RecX products. In addition to sperm motility, the E. coli strain ATCC 35218 also affected sperm viability, and induced caspase activity, oxidative stress and DNA fragmentation suggesting an interspecies variability in the amount and/or type of the produced spermatotoxic factors. In general, our results highlight the need for a careful evaluation of semen infections in the diagnostic process of the infertile man.

Membrane-Interacting DNA Nanotubes Induce Cancer Cell Death

DNA nanotechnology offers to build nanoscale structures with defined chemistries to precisely position biomolecules or drugs for selective cell targeting and drug delivery. Owing to the negatively charged nature of DNA, for delivery purposes, DNA is frequently conjugated with hydrophobic moieties, positively charged polymers/peptides and cell surface receptor-recognizing molecules or antibodies. Here, we designed and assembled cholesterol-modified DNA nanotubes to interact with cancer cells and conjugated them with cytochrome c to induce cancer cell apoptosis. By flow cytometry and confocal microscopy, we observed that DNA nanotubes efficiently bound to the plasma membrane as a function of the number of conjugated cholesterol moieties. The complex was taken up by the cells and localized to the endosomal compartment. Cholesterol-modified DNA nanotubes, but not unmodified ones, increased membrane permeability, caspase activation and cell death. Irreversible inhibition of caspase activity with a caspase inhibitor, however, only partially prevented cell death. Cytochrome c-conjugated DNA nanotubes were also efficiently taken up but did not increase the rate of cell death. These results demonstrate that cholesterol-modified DNA nanotubes induce cancer cell death associated with increased cell membrane permeability and are only partially dependent on caspase activity, consistent with a combined form of apoptotic and necrotic cell death. DNA nanotubes may be further developed as primary cytotoxic agents, or drug delivery vehicles, through cholesterol-mediated cellular membrane interactions and uptake.