Bleaching physiology: who's the 'weakest link' - host vs. symbiont?

Environmental stress, such as an increase in the sea surface temperature, triggers coral bleaching, a profound dysfunction of the mutualist symbiosis between the host cnidarians and their photosynthetic dinoflagellates of the Family Symbiodiniaceae. Because of climate change, mass coral bleaching events will increase in frequency and severity in the future, threatening the persistence of this iconic marine ecosystem at global scale. Strategies adapted to coral reefs preservation and restoration may stem from the identification of the succession of events and of the different molecular and cellular contributors to the bleaching phenomenon. To date, studies aiming to decipher the cellular cascade leading to temperature-related bleaching, emphasized the involvement of reactive species originating from compromised bioenergetic pathways (e.g. cellular respiration and photosynthesis). These molecules are responsible for damage to various cellular components causing the dysregulation of cellular homeostasis and the breakdown of symbiosis. In this review, we synthesize the current knowledge available in the literature on the cellular mechanisms caused by thermal stress, which can initiate or participate in the cell cascade leading to the loss of symbionts, with a particular emphasis on the role of each partner in the initiating processes.

Procedural learning as a measure of functional impairment in a mouse model of ischemic stroke.

Basal ganglia stroke is often associated with functional deficits in patients, including difficulties to learn and execute new motor skills (procedural learning). To measure procedural learning in a murine model of stroke (30min right MCAO), we submitted C57Bl/6J mice to various sensorimotor tests, then to an operant procedure (Serial Order Learning) specifically assessing the ability to learn a simple motor sequence. Results showed that MCAO affected the performance in some of the sensorimotor tests (accelerated rotating rod and amphetamine rotation test) and the way animals learned a motor sequence. The later finding seems to be caused by difficulties regarding the chunking of operant actions into a coherent motor sequence; the appeal for food rewards and ability to press levers appeared unaffected by MCAO. We conclude that assessment of motor learning in rodent models of stroke might improve the translational value of such models.

Assessment of behavioral flexibility after middle cerebral artery occlusion in mice.

Middle cerebral artery occlusion (MCAO) is the most common animal model of cerebral ischemia and induces various functional impairments. Long-lasting deficits resulting from MCAO however, remain insufficiently characterized, especially regarding cognition. Yet, behavioral flexibility, a prominent cognitive process is found impaired after stroke in humans. We thus used an operant-based task to assess behavioral flexibility in mice after MCAO. Three weeks after 30 min MCAO surgery, mice were subjected to a battery of sensorimotor tests (rotarod, vertical pole test, spontaneous locomotion and grip-strength test). Behavioral flexibility was then assessed in an operant task, in which mice, rewarded according to a FR5 schedule of reinforcement, had to alternate their operant responses between two levers from trial to trial. Regarding sensory and motor functioning, only the pole test yielded a significant difference between MCAO and sham mice. In the operant flexibility task, results showed a behavioral flexibility deficit in MCAO mice; neither the operant response acquisition nor the appeal for food rewards was altered. In conclusion, our operant-based task revealed a long-lasting behavioral flexibility deficit after MCAO in mice.

Increased cell proliferation and mucocyte density in the sea anemone Aiptasia pallida recovering from bleaching.

Recovery of coral after bleaching episodes is a critical period for the health of the reef ecosystem. While events such as symbiont (genus Symbiodinium) shifting/shuffling or tissue apoptosis have been demonstrated to occur following bleaching, little is known concerning tissue recovery or cell proliferation. Here, we studied the sea anemone Aiptasia pallida exposed to a transient elevation of water temperature combined with high illumination (33°C and 1900 µmol photons x m(-2) x s(-1) for 30 h). Following such treatment bleached anemones showed a significant reduction of their Symbiodinium density. Cell proliferation in the ectodermis and gastrodermis was determined by assessing the densities of cells labeled with a thymidine analogue (EdU). Cell proliferation significantly increased during the first day following stress in both tissue types. This increased cell proliferation returned to pre-stress values after one week. Although cell proliferation was higher in the ectodermis in absence of stress, it was relatively more pronounced in the gastrodermis of stressed anemones. In addition, the ratio of ectodermal mucocytes significantly increased three weeks after induced stress. These results suggest that thermal/photic stress coupled with the loss of the symbionts is able to enhance cell proliferation in both gastrodermis and ectodermis of cnidarians. While new cells formed in the gastrodermis are likely to host new Symbiodinium, the fate of new cells in the ectodermis was only partially revealed. Some new ectodermal cells may, in part, contribute to the increased number of mucocytes which could eventually help strengthen the heterotrophic state until restoration of the symbiosis.

A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons.

BACKGROUND: Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation. RESULTS: To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y). CONCLUSIONS: Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the Dcx coding sequence are sufficient to induce specific Dcx expression in vitro: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. Furthermore, a region in the 3'-end region of the Dcx promoter is highly conserved across several species and exerts positive control on Dcx transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse Dcx regulatory sequence is essential for Dcx gene expression during differentiation of neuronal precursors.

Anxiety in adult female mice following perinatal exposure to chlorpyrifos.

Epidemiologic studies suggested a possible link between prenatal exposure to organophosphate insecticides (OP) and long-term mental delay and some behavioral troubles. Experimental studies in rats and mice have confirmed that a relatively short exposure to low doses of OP such as chlorpyrifos (CPF) during specific perinatal periods decreased anxiety-like behaviors. In the present study, we report that chronic perinatal exposure (GD15-PND14) to low doses of CPF leads to an increase (and not a decrease) in anxiety-like behaviors of female mouse offspring. Pregnant or lactating female mice were exposed to CPF (0.2; 1; or 5 mg/kg day) by oral treatment during 18 consecutive days. Following a recovery period of several weeks, the anxiety of adult female offspring was determined using neurobehavioral tests (elevated plus-maze and light/dark box tests). Our results showed that CPF-exposed female offspring were more anxious than controls. In addition, the magnitude of anxiety profile alterations depended on the level of exposure to CPF during gestation and lactation with a maximal effect observed at the 1 mg/kg day dose. Our results confirm that OP exposure during the perinatal period can induce long-term alterations in mouse anxiety-like behaviors and suggest that the routes of administration and the duration of OP exposure during brain development may be factors to consider when studying the development of anxiety.

The usefulness of operant conditioning procedures to assess long-lasting deficits following transient focal ischemia in mice.

In this study, we examined a number of short and long-term sensorimotor, behavioural and cognitive consequences of an experimental ischemia induced by a 60-min right middle cerebral artery occlusion (MCAO) in 129S2 mice. During 14 days after surgery, a classical sensorimotor assessment was conducted using hanging wire test, negative geotaxis test, grip strength test, accelerated rotarod test and locomotor activity-meter. In order to provide a technique for the assessment of more resistant consequences of ischemia on fine psychomotor control, the peak procedure (a modified version of the operant fixed-interval schedule of reinforcement) was used. This procedure also helped to objectify temporal perception in mice five weeks following surgery. On several sensorimotor tests, ischemic mice showed some degree of impairment which rapidly tended to improve after stroke, a profile of results substantially consistent with previous studies. Five weeks post-surgery, ischemic mice tested with the peak procedure exhibited a moderate but yet significant temporal regulation impairment along with a reduced response rate compared to control mice. The present results suggest that the peak procedure and other derived operant schedules of reinforcement may provide useful and sensitive tools for the long-term assessment of both behavioural and cognitive aspects of the consequences of an experimental ischemia.

Subchronic alpha-linolenic acid treatment enhances brain plasticity and exerts an antidepressant effect: a versatile potential therapy for stroke.

Omega-3 polyunsaturated fatty acids are known to have therapeutic potential in several neurological and psychiatric disorders. However, the molecular mechanisms of action underlying these effects are not well elucidated. We previously showed that alpha-linolenic acid (ALA) reduced ischemic brain damage after a single treatment. To follow-up this finding, we investigated whether subchronic ALA treatment promoted neuronal plasticity. Three sequential injections with a neuroprotective dose of ALA increased neurogenesis and expression of key proteins involved in synaptic functions, namely, synaptophysin-1, VAMP-2, and SNAP-25, as well as proteins supporting glutamatergic neurotransmission, namely, V-GLUT1 and V-GLUT2. These effects were correlated with an increase in brain-derived neurotrophic factor (BDNF) protein levels, both in vitro using neural stem cells and hippocampal cultures and in vivo, after subchronic ALA treatment. Given that BDNF has antidepressant activity, this led us to test whether subchronic ALA treatment could produce antidepressant-like behavior. ALA-treated mice had significantly reduced measures of depressive-like behavior compared with vehicle-treated animals, suggesting another aspect of ALA treatment that could stimulate functional stroke recovery by potentially combining acute neuroprotection with long-term repair/compensatory plasticity. Indeed, three sequential injections of ALA enhanced protection, either as a pretreatment, wherein it reduced post-ischemic infarct volume 24 h after a 1-hour occlusion of the middle cerebral artery or as post-treatment therapy, wherein it augmented animal survival rates by threefold 10 days after ischemia.

Benfotiamine, a synthetic S-acyl thiamine derivative, has different mechanisms of action and a different pharmacological profile than lipid-soluble thiamine disulfide derivatives.

BACKGROUND: Lipid-soluble thiamine precursors have a much higher bioavailability than genuine thiamine and therefore are more suitable for therapeutic purposes. Benfotiamine (S-benzoylthiamine O-monophosphate), an amphiphilic S-acyl thiamine derivative, prevents the progression of diabetic complications, probably by increasing tissue levels of thiamine diphosphate and so enhancing transketolase activity. As the brain is particularly sensitive to thiamine deficiency, we wanted to test whether intracellular thiamine and thiamine phosphate levels are increased in the brain after oral benfotiamine administration. RESULTS: Benfotiamine that is practically insoluble in water, organic solvents or oil was solubilized in 200 mM hydroxypropyl-beta-cyclodextrin and the mice received a single oral administration of 100 mg/kg. Though thiamine levels rapidly increased in blood and liver to reach a maximum after one or two hours, no significant increase was observed in the brain. When mice received a daily oral administration of benfotiamine for 14 days, thiamine derivatives were increased significantly in the liver but not in the brain, compared to control mice. In addition, incubation of cultured neuroblastoma cells with 10 muM benfotiamine did not lead to increased intracellular thiamine levels. Moreover, in thiamine-depleted neuroblastoma cells, intracellular thiamine contents increased more rapidly after addition of thiamine to the culture medium than after addition of benfotiamine for which a lag period was observed. CONCLUSION: Our results show that, though benfotiamine strongly increases thiamine levels in blood and liver, it has no significant effect in the brain. This would explain why beneficial effects of benfotiamine have only been observed in peripheral tissues, while sulbutiamine, a lipid-soluble thiamine disulfide derivative, that increases thiamine derivatives in the brain as well as in cultured cells, acts as a central nervous system drug. We propose that benfotiamine only penetrates the cells after dephosphorylation by intestinal alkaline phosphatases. It then enters the bloodstream as S-benzoylthiamine that is converted to thiamine in erythrocytes and in the liver. Benfotiamine, an S-acyl derivative practically insoluble in organic solvents, should therefore be differentiated from truly lipid-soluble thiamine disulfide derivatives (allithiamine and the synthetic sulbutiamine and fursultiamine) with a different mechanism of absorption and different pharmacological properties.

Effects of hypoxia on stress proteins in the piglet brain at birth.

Newborn piglets were submitted to normobaric hypoxia (5% O2, 95% N2) for either 1 or 4 h. The effects of hypoxia on the neonatal brain were characterized through a time-course analysis of levels of various proteins such as heat shock proteins (HSP27, 70, and 90), hypoxia inducible factor-1alpha (HIF-1alpha), neuronal nitric oxide synthase (nNOS), hemeoxygenase-2 (HO-2), and caspase-3. The expression of these proteins was determined at different stages of recovery up to 72 h in cerebellum, cortex, and hippocampus by Western blot analysis in hypoxic maintained animals that were made hypoxic at either 20 or 37 degrees C. In all regions of the brain, HIF-1alpha and HSP27 expression were strongly increased until 22 h of recovery. No significant changes were observed for HSP70, HSP90, and HO-2. A small elevation of expression of nNOS was observed at early stages in the cerebellum and the cortex with no change in the hippocampus. Expression of caspase 3 was strongly increased in the cortex 24 and 48 h after hypoxia but unchanged in the hippocampus. These results are presented in terms of the porcine model of nonischemic hypoxia and its delayed neuronal effects on the cerebral outcome. Because of their recently established biochemical and functional interactions, the expression of the main HSPs, HIF-1alpha, nNOS, and caspase-3 after hypoxia are delineated.

EGF amplifies the replacement of parvalbumin-expressing striatal interneurons after ischemia.

EGF promotes proliferation and migration of stem/progenitor cells in the normal adult brain. The effect of epidermal growth factor on neurogenesis in ischemic brain is unknown, however. Here we show that intraventricular administration of EGF and albumin augments 100-fold neuronal replacement in the injured adult mouse striatum after cerebral ischemia. Newly born immature neurons migrate into the ischemic lesion and differentiate into mature parvalbumin-expressing neurons, replacing more than 20% of the interneurons lost by 13 weeks after ischemia and representing 2% of the total BrdU-labeled cells. These data suggest that administration of EGF and albumin could be used to manipulate endogenous neurogenesis in the injured brain and to promote brain self-repair.

Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids.

Many cellular responses to corticosteroids involve the transcriptional modulation of target genes by the glucocorticoid receptor (GR). A rapid, non-nuclear effect of GR was found to mediate neuroprotection. High-dose corticosteroids (20 mg/kg intraperitoneally), given within 2 hours of transient cerebral ischemia, acutely increased endothelial nitric oxide synthase (eNOS) activity, augmented regional cerebral blood flow (CBF) by 40% to 50%, and reduced cerebral infarct size by 32%. These neuroprotective effects of corticosteroids were abolished by the GR antagonist RU486 and by inhibition of phosphatidylinositol 3-kinase (PI3K), and were absent in eNOS(-/-) mice. To determine the mechanism by which GR activated eNOS, we measured the effect of corticosteroids on PI3K and the protein kinase Akt. In a ligand-dependent manner, GR activated PI3K and Akt in vitro and in vivo caused NO-dependent vasodilation, which was blocked by cotreatment with RU486 or the PI3K inhibitor LY294002 but not by transcriptional inhibitors. Indeed, a mutant GR, which cannot dimerize and bind to DNA, still activated PI3K and Akt in response to corticosteroids. These findings indicate that non-nuclear GR rapidly activates eNOS through the PI3K/Akt pathway and suggest that this mechanism mediates the acute neuroprotective effects of corticosteroids through augmentation of CBF.

Heat shock transcription factor 2 is not essential for embryonic development, fertility, or adult cognitive and psychomotor function in mice.

Members of the heat shock factor (HSF) family are evolutionarily conserved regulators that share a highly homologous DNA-binding domain. In mammals, HSF1 is the main factor controlling the stress-inducible expression of Hsp genes while the functions of HSF2 and HSF4 are less clear. Based on its developmental profile of expression, it was hypothesized that HSF2 may play an essential role in brain and heart development, spermatogenesis, and erythroid differentiation. To directly assess this hypothesis and better understand the underlying mechanisms that require HSF2, we generated Hsf2 knockout mice. Here, we report that Hsf2(-/-) mice are viable and fertile and exhibit normal life span and behavioral functions. We conclude that HSF2, most probably because its physiological roles are integrated into a redundant network of gene regulation and function, is dispensable for normal development, fertility, and postnatal psychomotor function.

Acute cardiovascular protective effects of corticosteroids are mediated by non-transcriptional activation of endothelial nitric oxide synthase.

Corticosteroids have been shown to exert beneficial effects in the treatment of acute myocardial infarction, but the precise mechanisms underlying their protective effects are unknown. Here we show that high-dose corticosteroids exert cardiovascular protection through a novel mechanism involving the rapid, non-transcriptional activation of endothelial nitric oxide synthase (eNOS). Binding of corticosteroids to the glucocorticoid receptor (GR) stimulated phosphatidylinositol 3-kinase and protein kinase Akt, leading to eNOS activation and nitric oxide dependent vasorelaxation. Acute administration of pharmacological concentrations of corticosteroids in mice led to decreased vascular inflammation and reduced myocardial infarct size following ischemia and reperfusion injury. These beneficial effects of corticosteroids were abolished by GR antagonists or eNOS inhibitors in wild-type mice and were completely absent in eNOS-deficient (Nos3(-/-)) mice. The rapid activation of eNOS by the non-nuclear actions of GR, therefore, represents an important cardiovascular protective effect of acute high-dose corticosteroid therapy.