Stress-induced Death Research Articles

A selective inhibitor of mitofusin 1-βIIPKC association improves heart failure outcome in rats

We previously demonstrated that beta II protein kinase C (βIIPKC) activity is elevated in failing hearts and contributes to this pathology. Here we report that βIIPKC accumulates on the mitochondrial outer membrane and phosphorylates mitofusin 1 (Mfn1) at serine 86. Mfn1 phosphorylation results in partial loss of its GTPase activity and in a buildup of fragmented and dysfunctional mitochondria in heart failure. βIIPKC siRNA or a βIIPKC inhibitor mitigates mitochondrial fragmentation and cell death. We confirm that Mfn1-βIIPKC interaction alone is critical in inhibiting mitochondrial function and cardiac myocyte viability using SAMβA, a rationally-designed peptide that selectively antagonizes Mfn1-βIIPKC association. SAMβA treatment protects cultured neonatal and adult cardiac myocytes, but not Mfn1 knockout cells, from stress-induced death. Importantly, SAMβA treatment re-establishes mitochondrial morphology and function and improves cardiac contractility in rats with heart failure, suggesting that SAMβA may be a potential treatment for patients with heart failure.

酸化ストレス誘発されるニューロン障害およびミクログリア依存性脳炎症に対するRatanasampilの抑制作用

Generation of reactive oxygen species (ROS) causes lipids, proteins and DNA damage, resulting in neuronal damage and neruoinflammation. Ratanasampil (RNSP), a traditional Tibetan medicine, clinically used for the mild-to moderate AD patients living at high altitude. In vivo, RNSP improved the learning and memory in an AD mouse model (Tg2576). However, mechanism underlying the effects of RNSP is unknown. In SH-SY5Y cells, RNSP significantly ameliorated the H2O2 –induced cytotoxicity. Furthermore, RNSP significantly reduced the H2O2-induced 8-oxo-2′-deoxyguanosine and attenuated the phosphorylation of p38 and ERK 1/2. In MG6 microglia, RNSP significantly ameliorated the cytotoxicity induced by hypoxia-reoxygenation. Furthermore, RNSP significantly suppressed the H6/R24-induced pro-inflammatory cytokines, ROS, DNA damage and phosphorylation of IκBα. These observations suggest that RNSP suppressed the H2O2-induced neuronal death through downregulation of p38-ERK activation and regulated the H/R-induced neuroinflammation through inhibition of oxidative stress and the activation of NFκB in MG6 cells. Therefore, RNSP may be beneficial for preventing oxidative stress-induced neuronal death and neuroinflammation.

KEAP1/NRF2 axis regulates H2O2-induced apoptosis of pancreatic β-cells

In human pancreatic β-cells, oxidative stress and cellular injures can be induced by H2O2 treatment. The KEAP1/NRF2 axis is a key antioxidant signaling pathway. The present study attempted to elucidate the mechanism by which the KEAP1/NRF2 axis mediates oxidative stress-induced death in pancreatic β-cells. Our data showed that H2O2 treatment obviously induced the apoptosis of β-cells. Further experiments demonstrated that KEAP1 expression was downregulated in H2O2-treated pancreatic β-cells and this change correlated with increase in the cellular abundance and nuclear translocation of NRF2. The restoration of KEAP1 expression in cells resulted in a recovery of cell proliferation and inhibition of apoptosis. Furthermore, we found that KEAP1 overexpression negatively regulated the abundance of NRF2, subsequently causing decreased antioxidant response element activation. This led to HO-1 protein downregulation in H2O2-treated human pancreatic β-cells, which was also observed in NRF2-silenced β-cells. Conversely, the silencing of KEAP1 led to NRF2 upregulation and inhibited ARE and HO-1 signaling in pancreatic β-cells. The increase in the abundance of NRF2 following treatment with H2O2 drastically elevated the production of BAX, FAS, FAS-L, CASP-3, and CASP-9, and this change was reversed by KEAP1 overexpression or NRF2 silencing. Taken together, H2O2 treatment activated KEAP1/NRF2 signaling to promote the production of pro-apoptotic factors and consequently led to the apoptosis of human pancreatic β-cells.

JUN and ATF3 Regulate the Transcriptional Output of the Unfolded Protein Response to Support Acute Myeloid Leukemia

The genomic alterations that drive acute myeloid leukemia (AML) are well established, however, the molecular pathways that are deregulated as a consequence of these mutations and their impact on AML development and progression remain poorly understood. We and others have previously shown that the transcription factor (TF) JUN, though not mutated, is highly expressed in multiple AML sub-types (e.g. normal karyotype (NK-AML), complex karyotype and 11q23 rearrangements) relative to healthy hematopoietic stem and progenitor cells (HSPCs). We also recently reported that JUN supports cell survival in a panel of genetically diverse AML cell lines and patient-derived AML samples ex vivo. Moreover, we showed that JUN inhibition significantly delays disease onset and improves survival in a genetically engineered mouse model (GEMM) of AML driven by MLL-AF9. Of critical importance, we have now observed that deletion of JUN does not negatively impact steady-state hematopoiesis or the hematopoietic reconstituting potential of HSPCs. While these results position JUN as an attractive therapeutic target in AML, previous efforts to develop JUN inhibitors have been unsuccessful. Therefore, our objective is to elucidate the mechanisms by which JUN supports AML in order to identify putative therapeutic targets in this pathway.JUN regulates many transcriptional programs by forming hetero-dimers with one of many other TFs and our goal was to identify which dimeric partner cooperates with JUN to regulate transcriptional programs that support AML. We previously reported that JUN supports AML, in part, by regulating the transcriptional output of the unfolded protein response (UPR), which is a signal transduction network that helps cells negotiate endoplasmic reticulum (ER) stress. To identify dimerization partners that cooperate with JUN to drive transcriptional output of the UPR, we utilized the UCSC genome browser, which contains the chromatin immunoprecipitation-sequencing (ChIP-seq) data for JUN as well as 15 known dimerization partners of JUN. Using this database, we found that JUN localizes to the promoters of 35 UPR genes. Notably, we found that the stress-response factor ATF3 co-localizes with JUN at 31 of these 35 promoters (88.6%), exceeding the co-localization of the other 14 factors. Consistent with these observations, we found that shRNA-mediated inhibition of either JUN or ATF3 leads to decreased mRNA expression of the UPR targets genes (XBP1s, ATF4, DDIT3, HSPA5, GRP94, ATF6, EIF2AK3and ERN1) in human AML cell lines (e.g. THP-1, OCI-AML3, U937, MOLM14) as well as leukemia cells derived from the MLL-AF9-driven GEMM of AML. We also observed that inhibition of either JUN or ATF3 blocked the induction of UPR signaling and rendered human and mouse AML cells more susceptible to ER stress-induced death further suggesting that JUN and ATF3 regulate the transcriptional output of the UPR.To determine if ATF3 expression, like JUN, is deregulated in human AML, we performed a retrospective analysis of AML patient gene expression profiles and found that elevated expression of ATF3 is significantly associated with worse outcomes in two distinct datasets from NK-AML patients (p=0.0248, p=0.0049). Furthermore, we found that ATF3 expression is higher in AMLs with FLT3-ITD or 11q23 rearrangements, compared to healthy HSPCs. Based on these data, we investigated the functional role of ATF3 in AML. First, we found that shRNA-mediated inhibition of ATF3 significantly reduced the colony forming capacity of mouse AML cells expressing MLL-AF9 but, importantly, not healthy HSPCs. Furthermore, we found that ATF3 inhibition impeded the growth and survival of AML cells from a second GEMM of AML driven by the deletion of Dnmt3a and Tet2 in combination with expression of FLT3-ITD. We also observed that, similar to JUN inhibition, shRNA-mediated inhibition of ATF3 significantly reduced disease burden in MLL-AF9-driven AML in vivo (p=0.0028). At the cellular level, we found that AML cells depleted of ATF3 displayed increased CD11b expression and annexin V staining suggesting that ATF3 may support the differentiation blockade in AML.Collectively, our data suggest that JUN and ATF3 cooperate to support a variety of AML subtypes by regulating transcriptional programs, such as the UPR and our current efforts are focused on identifying additional targetable JUN/ATF3-regulated transcriptional programs that support AML. DisclosuresNo relevant conflicts of interest to declare.

Laminar Flow Inhibits ER Stress-Induced Endothelial Apoptosis through PI3K/Akt-Dependent Signaling Pathway.

Atherosclerosis preferentially involves in prone area of low and disturbed blood flow while steady and high levels of laminar blood flow are relatively protected from atherosclerosis. Disturbed flow induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). ER stress is caused under stress that disturbs the processing and folding of proteins resulting in the accumulation of misfolded proteins in the ER and activation of the UPR. Prolonged or severe UPR leads to activate apoptotic signaling. Recent studies have indicated that disturbed flow significantly up-regulated p-ATF6α, p-IRE1α, and its target spliced XBP-1. However, the role of laminar flow in ER stress-mediated endothelial apoptosis has not been reported yet. The present study thus investigated the role of laminar flow in ER stress-dependent endothelial cell death. The results demonstrated that laminar flow protects ER stress-induced cleavage forms of PARP-1 and caspase-3. Also, laminar flow inhibits ER stress-induced p-eIF2α, ATF4, CHOP, spliced XBP-1, ATF6 and JNK pathway; these effects are abrogated by pharmacological inhibition of PI3K with wortmannin. Finally, nitric oxide affects thapsigargin-induced cell death in response to laminar flow but not UPR. Taken together, these findings indicate that laminar flow inhibits UPR and ER stress-induced endothelial cell death via PI3K/Akt pathway.

NF-\u03baB p65 directs sex-specific neuroprotection in human neurons

Protection of neurons against oxidative stress is crucial during neuronal development, maintenance and for treating neurodegenerative diseases. However, little is known about the molecular mechanisms underlying sex-specific maturation and survival of neurons. In the present study, we demonstrate NF-κB-p65 mediated neuroprotection in human glutamatergic neurons differentiated from inferior turbinate stem cells (ITSCs) in a sex-dependent manner. We successfully differentiated ITSCs into MAP-2+/NF200+/Synaptophysin+/vGlut2+-glutamatergic neurons in vitro and ex vivo and validated their functionality. TNF-α-dependent NF-κB-p65 activation was accompanied by significant neuroprotection against oxidative stress-induced neuronal death, which was surprisingly higher in neurons from female donors. Accordingly, sex-specific neuroprotection of female neurons was followed by an increased expression of special NF-κB target genes SOD2 and IGF2. Among these, SOD2 is a well known gene protecting cells against oxidative stress resulting in longevity. In addition, IGF2 is known to promote synapse formation and spine maturation, and it has antioxidant and neuroprotective effects against oxidative damage. In conclusion, we show that NF-κB-p65 is a key player in neuroprotection of human neurons, however the protective gene expression program beneath it differs between sexes. Our findings are in accordance with the increasing evidences pointing towards sex-specific differences in risk and severity of neurodegenerative diseases.

Rabbitpox in New Zealand White Rabbits: A Therapeutic Model for Evaluation of Poxvirus Medical Countermeasures Under the FDA Animal Rule.

The elimination of smallpox as an endemic disease and the obvious ethical problems with clinical challenge requires the efficacy evaluation of medical countermeasures against smallpox using the FDA Animal Rule. This approach requires the evaluation of antiviral efficacy in an animal model whose infection recapitulates the human disease sufficiently well enough to provide predictive value of countermeasure effectiveness. The narrow host range of variola virus meant that no other animal species was sufficiently susceptible to variola to manifest a disease with predictive value. To address this dilemma, the FDA, after a public forum with virologists in December 2011, suggested the development of two animal models infected with the cognate orthopoxvirus, intradermal infection of rabbits and intranasal infection of mice, to supplement the non-human primate models in use. In this manuscript, we describe the development of an intradermal challenge model of New Zealand White rabbits with rabbitpox virus (RPXV) for poxvirus countermeasure evaluation. Lethality of RPXV was demonstrated in both 9 and 16-weeks old rabbits with an LD50 < 10 PFU. The natural history of RPXV infection was documented in both ages of rabbits by monitoring the time to onset of abnormal values in clinical data at a lethal challenge of 300 PFU. All infected animals became viremic, developed a fever, exhibited weight loss, developed secondary lesions, and were euthanized after 7 or 8 days. The 16-weeks RPXV-infected animals exhibiting similar clinical signs with euthanasia applied about a day later than for 9-weeks old rabbits. For all animals, the first two unambiguous indicators of infection were detection of viral copies by quantitative polymerase chain reaction and fever at 2 and 3 days following challenge, respectively. These biomarkers provide clinically-relevant trigger(s) for initiating therapy. The major advantage for using 16-weeks NZW rabbits is that older rabbits were more robust and less subject to stress-induced death allowing more reproducible studies.

Cyclin-dependent kinase 5-mediated phosphorylation of chloride intracellular channel 4 promotes oxidative stress-induced neuronal death

Oxidative stress can cause apoptosis in neurons and may result in neurodegenerative diseases. However, the signaling mechanisms leading to oxidative stress–induced neuronal apoptosis are not fully understood. Oxidative stress stimulates aberrant activation of cyclin-dependent kinase 5 (CDK5), thought to promote neuronal apoptosis by phosphorylating many cell death-related substrates. Here, using protein pulldown methods, immunofluorescence experiments and in vitro kinase assays, we identified chloride intracellular channel 4 (CLIC4), the expression of which increases during neuronal apoptosis, as a CDK5 substrate. We found that activated CDK5 phosphorylated serine 108 in CLIC4, increasing CLIC4 protein stability, and accumulation. Pharmacological inhibition or shRNA-mediated silencing of CDK5 decreased CLIC4 levels in neurons. Moreover, CLIC4 overexpression led to neuronal apoptosis, whereas knockdown or pharmacological inhibition of CLIC4 attenuated H2O2-induced neuronal apoptosis. These results implied that CLIC4, by acting as a substrate of CDK5, mediated neuronal apoptosis induced by aberrant CDK5 activation. Targeting CLIC4 in neurons may therefore provide a therapeutic approach for managing progressive neurodegenerative diseases that arise from neuronal apoptosis.

Heart specific knockout of Ndufs4 ameliorates ischemia reperfusion injury

RationaleIschemic heart disease (IHD) is a leading cause of mortality. The most effective intervention for IHD is reperfusion, which ironically causes ischemia reperfusion (I/R) injury mainly due to oxidative stress-induced cardiomyocyte death. The exact mechanism and site of reactive oxygen species (ROS) generation during I/R injury remain elusive. ObjectiveWe aim to test the hypothesis that Complex I-mediated forward and reverse electron flows are the major source of ROS in I/R injury of the heart. Methods and resultsWe used a genetic model of mitochondrial Complex I deficiency, in which a Complex I assembling subunit, Ndufs4 was knocked out in the heart (Ndufs4H−/−). The Langendorff perfused Ndufs4H−/− hearts exhibited significantly reduced infarct size (45.3 ± 5.5% in wild type vs 20.9 ± 8.1% in Ndufs4H−/−), recovered contractile function, and maintained mitochondrial membrane potential after no flow ischemia and subsequent reperfusion. In cultured adult cardiomyocytes from Ndufs4H−/− mice, I/R mimetic treatments caused minimal cell death. Reintroducing Ndufs4 in Ndufs4H−/− cardiomyocytes abolished the protection. Mitochondrial NADH declined much slower in Ndufs4H−/− cardiomyocytes during reperfusion suggesting decreased forward electron flow. Mitochondrial flashes, a marker for mitochondrial respiration, were inhibited in Ndufs4H−/− cardiomyocytes at baseline and during I/R, which was accompanied by preserved aconitase activity suggesting lack of oxidative damage. Finally, pharmacological blockade of forward and reverse electron flow at Complex I inhibited I/R-induced cell death. ConclusionsThese results provide the first genetic evidence supporting the central role of mitochondrial Complex I in I/R injury of mouse heart. The study also suggests that both forward and reverse electron flows underlie oxidative cardiomyocyte death during reperfusion.

Altered cellular redox homeostasis and redox responses under standard oxygen cell culture conditions versus physioxia

In vivo, mammalian cells reside in an environment of 0.5–10% O2 (depending on the tissue location within the body), whilst standard in vitro cell culture is carried out under room air. Little is known about the effects of this hyperoxic environment on treatment-induced oxidative stress, relative to a physiological oxygen environment. In the present study we investigated the effects of long-term culture under hyperoxia (air) on photodynamic treatment. Upon photodynamic irradiation, cells which had been cultured long-term under hyperoxia generated higher concentrations of mitochondrial reactive oxygen species, compared with cells in a physioxic (2% O2) environment. However, there was no significant difference in viability between hyperoxic and physioxic cells. The expression of genes encoding key redox homeostasis proteins and the activity of key antioxidant enzymes was significantly higher after the long-term culture of hyperoxic cells compared with physioxic cells. The induction of antioxidant genes and increased antioxidant enzyme activity appear to contribute to the development of a phenotype that is resistant to oxidative stress-induced cellular damage and death when using standard cell culture conditions. The results from experiments using selective inhibitors suggested that the thioredoxin antioxidant system contributes to this phenotype. To avoid artefactual results, in vitro cellular responses should be studied in mammalian cells that have been cultured under physioxia. This investigation provides new insights into the effects of physioxic cell culture on a model of a clinically relevant photodynamic treatment and the associated cellular pathways.

Hippo Signaling Pathway Dysregulation in Human Huntington\u2019s Disease Brain and Neuronal Stem Cells

The Hippo signaling pathway is involved in organ size regulation and tumor suppression. Although inhibition of Hippo leads to tumorigenesis, activation of Hippo may play a role in neurodegeneration. Specifically, activation of the upstream regulator, mammalian sterile 20 (STE20)-like kinase 1 (MST1), reduces activity of the transcriptional co-activator Yes-Associated Protein (YAP), thereby mediating oxidative stress-induced neuronal death. Here, we investigated the possible role of this pathway in Huntington’s disease (HD) pathogenesis. Our results demonstrate a significant increase in phosphorylated MST1, the active form, in post-mortem HD cortex and in the brains of CAG knock-in HdhQ111/Q111 mice. YAP nuclear localization was also decreased in HD post-mortem cortex and in neuronal stem cells derived from HD patients. Moreover, there was a significant increase in phosphorylated YAP, the inactive form, in HD post-mortem cortex and in HdhQ111/Q111 brain. In addition, YAP was found to interact with huntingtin (Htt) and the chaperone 14-3-3, however this interaction was not altered in the presence of mutant Htt. Lastly, YAP/TEAD interactions and expression of Hippo pathway genes were altered in HD. Together, these results demonstrate that activation of MST1 together with a decrease in nuclear YAP could significantly contribute to transcriptional dysregulation in HD.

Increased mitochondrial respiration promotes survival from endoplasmic reticulum stress.

Protein misfolding in the endoplasmic reticulum (ER) is accompanied by adaptive cellular responses to promote cell survival. We now show that activation of mitochondrial respiration is a critical component of an adaptive ER stress response, requiring the unfolded protein response (UPR) sensor Ire1, and also calcium signaling via calcineurin. In yeast and mammalian cells lacking Ire1 or calcineurin, respiratory activation is impaired in response to ER stress; accumulation of mitochondrial reactive oxygen species (ROS) triggers cell death as abrogation of ROS by antioxidants or loss of the electron transport chain (in yeast) can rescue cells from death. Significantly, cells are rescued from ER stress-induced death by mitochondrial uncoupling by CCCP to increase O2 consumption (and increase the efficiency of electron transfer). Remarkably, genetic and pharmacologic strategies to promote mitochondrial biogenesis and increase O2 consumption also alleviate ER stress-mediated ROS and death in yeast and mammalian cells. Moreover, in a yeast genetic screen, three mitochondrial proteins Mrx9, Mrm1, and Aim19 that increase mitochondrial biogenesis were identified as high copy suppressors of ER stress-mediated cell death. Our results show that enhanced mitochondrial biogenesis, linked to improved efficiency of the electron transport chain, is a powerful strategy to block ROS accumulation and promote cell survival during ER stress in eukaryotic cells.

A genetic program mediates cold-warming response and promotes stress-induced phenoptosis in C. elegans.

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming exposure. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild-population kin selection under resource-limiting and thermal stress conditions.

CacyBP/SIP, a Hsp90 binding chaperone, in cellular stress response.

CacyBP/SIP interacts with Hsp90 and is able to protect proteins from denaturation and/or aggregation induced by elevated temperature. In this work we studied the influence of different stress factors on CacyBP/SIP level in HEp-2 cells. We have found that H2O2 and radicicol treatment resulted in a significant increase (up to 40%) in the CacyBP/SIP level. We have also found that HEp-2 cells overexpressing CacyBP/SIP were more resistant to stress-induced death. Further studies have revealed that the Hsf1 transcription factor binds to the CacyBP/SIP gene promoter and up-regulates CacyBP/SIP expression under stress conditions. To check whether the CacyBP/SIP protein might play a role in stress responses in vivo, we analyzed its level in selected brain structures of control and stressed mice. We have found that the level of the CacyBP/SIP protein was higher in the thalamus/hypothalamus, hippocampus and brainstem of stressed mice. Thus, the presented results clearly indicate that CacyBP/SIP is involved in cellular stress response.

Expression of Carbonic Anhydrase III, a Nucleus Pulposus Phenotypic Marker, is Hypoxia-responsive and Confers Protection from Oxidative Stress-induced Cell Death

The integrity of the avascular nucleus pulposus (NP) phenotype plays a crucial role in the maintenance of intervertebral disc health. While advances have been made to define the molecular phenotype of healthy NP cells, the functional relevance of several of these markers remains unknown. In this study, we test the hypothesis that expression of Carbonic Anhydrase III (CAIII), a marker of the notochordal NP, is hypoxia-responsive and functions as a potent antioxidant without a significant contribution to pH homeostasis. NP, but not annulus fibrosus or end-plate cells, robustly expressed CAIII protein in skeletally mature animals. Although CAIII expression was hypoxia-inducible, we did not observe binding of HIF-1α to select hypoxia-responsive-elements on Car3 promoter using genomic chromatin-immunoprecipitation. Similarly, analysis of discs from NP-specific HIF-1α null mice suggested that CAIII expression was independent of HIF-1α. Noteworthy, silencing CAIII in NP cells had no effect on extracellular acidification rate, CO2 oxidation rate, or intracellular pH, but rather sensitized cells to oxidative stress-induced death mediated through caspase-3. Our data clearly suggests that CAIII serves as an important antioxidant critical in protecting NP cells against oxidative stress-induced injury.

The mTOR-S6 kinase pathway promotes stress granule assembly

Stress granules are cytoplasmic mRNA-protein complexes that form upon the inhibition of translation initiation and promote cell survival in response to environmental insults. However, they are often associated with pathologies, including neurodegeneration and cancer, and changes in their dynamics are implicated in ageing. Here we show that the mTOR effector kinases S6 kinase 1 (S6K1) and S6 kinase 2 (S6K2) localise to stress granules in human cells and are required for their assembly and maintenance after mild oxidative stress. The roles of S6K1 and S6K2 are distinct, with S6K1 having a more significant role in the formation of stress granules via the regulation of eIF2α phosphorylation, while S6K2 is important for their persistence. In C. elegans, the S6 kinase orthologue RSKS-1 promotes the assembly of stress granules and its loss of function sensitises the nematodes to stress-induced death. This study identifies S6 kinases as regulators of stress granule dynamics and provides a novel link between mTOR signalling, translation inhibition and survival.

Transcriptome analysis of embryonic domains in Norway spruce reveals potential regulators of suspensor cell death.

The terminal differentiation and elimination of the embryo-suspensor is the earliest manifestation of programmed cell death (PCD) during plant ontogenesis. Molecular regulation of suspensor PCD remains poorly understood. Norway spruce (Picea abies) embryos provide a powerful model for studying embryo development because of their large size, sequenced genome, and the possibility to obtain a large number of embryos at a specific developmental stage through somatic embryogenesis. Here, we have carried out global gene expression analysis of the Norway spruce embryo-suspensor versus embryonal mass (a gymnosperm analogue of embryo proper) using RNA sequencing. We have identified that suspensors have enhanced expression of the NAC domain-containing transcription factors, XND1 and ANAC075, previously shown to be involved in the initiation of developmental PCD in Arabidiopsis. The analysis has also revealed enhanced expression of Norway spruce homologues of the known executioners of both developmental and stress-induced cell deaths, such as metacaspase 9 (MC9), cysteine endopeptidase-1 (CEP1) and ribonuclease 3 (RNS3). Interestingly, a spruce homologue of bax inhibitor-1 (PaBI-1, for Picea abies BI-1), an evolutionarily conserved cell death suppressor, was likewise up-regulated in the embryo-suspensor. Since Arabidopsis BI-1 so far has been implicated only in the endoplasmic reticulum (ER)-stress induced cell death, we investigated its role in embryogenesis and suspensor PCD using RNA interference (RNAi). We have found that PaBI-1-deficient lines formed a large number of abnormal embryos with suppressed suspensor elongation and disturbed polarity. Cytochemical staining of suspensor cells has revealed that PaBI-1 deficiency suppresses vacuolar cell death and induces necrotic type of cell death previously shown to compromise embryo development. This study demonstrates that a large number of cell-death components are conserved between angiosperms and gymnosperms and establishes a new role for BI-1 in the progression of vacuolar cell death.

Evidence for effective structure-based neuromodulatory effects of new analogues of neurosteroid allopregnanolone.

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-pituitary-adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-proliferation-promoting effects, hamper the development of selective/targeted therapies. For example, although AP-induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-evoked cell-proliferation is contraindicated for steroid-sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-oxo-epi-AP), BR297 (O-allyl-epi-AP), BR351 (O-allyl-AP) and BR338 (12-oxo-AP). First, because AP is well-known as allosteric modulator of GABAA receptors (GABAA-R), we used the electrophysiological patch-clamp technique to determine the structure-activity relationship of our ANS on GABAA-activated current in NCB20 cells expressing functional GABAA-R. We found that the addition of 12-oxo-group did not significantly change the respective positive or negative allosteric effects of 3α-AP or 3β-(epi)-AP analogues. Importantly, substitution of the 3α-hydroxyl-group by 3α-O-allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-lasting desensitisation/inhibition of GABAA-R. Interestingly, replacement of the 3β-hydroxyl by 3β-O-allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-SY5Y neuronal cell viability/proliferation using MTT-reduction assays. Different dose-response curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-SY5Y cells against oxidative stress-induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

Glycogen synthase protects neurons from cytotoxicity of mutant huntingtin by enhancing the autophagy flux

Healthy neurons do not store glycogen while they do possess the machinery for the glycogen synthesis albeit at an inactive state. Neurons in the degenerating brain, however, are known to accumulate glycogen, although its significance was not well understood. Emerging reports present contrasting views on neuronal glycogen synthesis; a few reports demonstrate a neurotoxic effect of glycogen while a few others suggest glycogen to be neuroprotective. Thus, the specific role of glycogen and glycogen synthase in neuronal physiology is largely unexplored. Using cellular and animal models of Huntington’s disease, we show here that the overexpression of cytotoxic mutant huntingtin protein induces glycogen synthesis in the neurons by activating glycogen synthase and the overexpressed glycogen synthase protected neurons from the cytotoxicity of the mutant huntingtin. Exposure of neuronal cells to proteasomal blockade and oxidative stress also activate glycogen synthase to induce glycogen synthesis and to protect against stress-induced neuronal death. We show that the glycogen synthase plays an essential and inductive role in the neuronal autophagic flux, and helps in clearing the cytotoxic huntingtin aggregate. We also show that the increased neuronal glycogen inhibits the aggregation of mutant huntingtin, and thus could directly contribute to its clearance. Finally, we demonstrate that excessive autophagy flux is the molecular basis of cell death caused by the activation of glycogen synthase in unstressed neurons. Taken together, our results thus provide a novel function for glycogen synthase in proteolytic processes and offer insight into the role of glycogen synthase and glycogen in both survival and death of the neurons.

Neural deletion of Sh2b1 results in brain growth retardation and reactive aggression.

Psychiatric disorders are associated with aberrant brain development and/or aggressive behavior and are influenced by genetic factors; however, genes that affect brain aggression circuits remain elusive. Here, we show that neuronal Src-homology-2 (SH2)B adaptor protein-1 ( Sh2b1) is indispensable for both brain growth and protection against aggression. Global and brain-specific deletion of Sh2b1 decreased brain weight and increased aggressive behavior. Global and brain-specific Sh2b1 knockout (KO) mice exhibited fatal, intermale aggression. In a resident-intruder paradigm, latency to attack was markedly reduced, whereas the number and the duration of attacks was significantly increased in global and brain-specific Sh2b1 KO mice compared with wild-type littermates. Consistently, core aggression circuits were activated to a higher level in global and brain-specific Sh2b1 KO males, based on c-fos immunoreactivity in the amygdala and periaqueductal gray. Brain-specific restoration of Sh2b1 normalized brain size and reversed pathologic aggression and aberrant activation of core aggression circuits in Sh2b1 KO males. SH2B1 mutations in humans were linked to aberrant brain development and behavior. At the molecular level, Sh2b1 enhanced neurotrophin-stimulated neuronal differentiation and protected against oxidative stress-induced neuronal death. Our data suggest that neuronal Sh2b1 promotes brain development and the integrity of core aggression circuits, likely through enhancing neurotrophin signaling.-Jiang, L., Su, H., Keogh, J. M., Chen, Z., Henning, E., Wilkinson, P., Goodyer, I., Farooqi, I. S., Rui, L. Neural deletion of Sh2b1 results in brain growth retardation and reactive aggression.