An animal model of eating disorders associated with stressful experience in early life

Experience of childhood abuse is prevalent among patients with eating disorders, and dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis is implicated in its pathophysiology. Neonatal maternal separation is considered as an animal model of stressful experience early in life. Many of studies have demonstrated its impact both on the activity of HPA axis and the development of psycho-emotional disorders later in life. In this paper, a series of our researches on developing an animal model of eating disorders is reviewed. An animal model of neonatal maternal separation was used; Sprague-Dawley pups were separated from dam daily for 180 min during the first 2 weeks of life (MS) or undisturbed. Anxiety-/depression-like behaviors were observed in MS rats at the age of two months with decreased serotonergic activity in the hippocampus and the raphe. Post-weaning social isolation promoted food intake and weight gain of adolescent MS pups, with impacts on anxiety-like behaviors. Sustained hyperphagia was observed in the MS pups subjected to a fasting/refeeding cycle repeatedly during adolescence, with increased plasma corticosterone levels. Anhedonia, major symptom of depression, to palatable food was observed in adolescent MS pups with blunted response of the mesolimbic dopaminergic activity to stress. Results suggest that neonatal maternal separation lead to the development of eating disorders when it is challenged with social or metabolic stressors later in life, in which dysfunctions in the HPA axis and the brain monoaminergic systems may play important roles.     

Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.     

Cyclin-Dependent Kinase 5 Links Extracellular Cues to Actin Cytoskeleton During Dendritic Spine Development

Emerging evidence has indicated a regulatory role of cyclin-dependent kinase 5 (Cdk5) in synaptic plasticity as well as in higher brain functions, such as learning and memory. However, the molecular and cellular mechanisms underlying the actions of Cdk5 at synapses remain unclear. Recent findings demonstrate that Cdk5 regulates dendritic spine morphogenesis through modulating actin dynamics. Ephexin1 and WAVE-1, two important regulators of the actin cytoskeleton, have both been recently identified as substrates for Cdk5. Importantly, phosphorylation of these proteins by Cdk5 leads to dendritic spine loss, revealing a potential mechanism by which Cdk5 regulates synapse remodeling. Furthermore, Cdk5-dependent phosphorylation of ephexin1 is required for the ephrin-A1 mediated spine retraction, pointing to a critical role of Cdk5 in conveying signals from extracellular cues to actin cytoskeleton at synapses. Taken together, understanding the precise regulation of Cdk5 and its downstream targets at synapses would provide important insights into the multi-regulatory roles of Cdk5 in actin remodeling during dendritic spine development.Excitatory synaptic transmission occurs primarily at dendritic spines, small protrusions that extend from dendritic shafts. Emerging studies have shown that dendritic spines are dynamic structures which undergo changes in size, shape and number during development, and remain plastic in adult brain.¹ Regulation of spine morphology has been implicated to associate with changes of synaptic strength.² For example, enlargement and shrinkage of spines was reported to associate with certain forms of synaptic plasticity, i.e., long-term potentiation and long time depression, respectively.³ Thus, understanding the molecular mechanisms underlying the regulation of spine morphogenesis would provide insights into synapse development and plasticity. Receptor tyrosine kinases (RTKs) such as the Ephs are known to play critical roles in regulating spine morphogenesis. Eph receptors are comprised of 14 members, which are classified into EphAs and EphBs according to their sequence homology and ligand binding specificity. With a few exceptions, EphAs typically bind to A-type ligands, whereas EphBs bind to B-type ligands. During development of the central nervous system (CNS), ephrin-Eph interactions exert repulsive/attractive signaling, leading to regulation of axon guidance, topographic mapping and neural patterning.⁴ Activated Ephs trigger intracellular signaling cascades, which subsequently lead to remodeling of actin cytoskeleton through tyrosine phosphorylation of its target proteins or interaction with various cytoplasmic signaling proteins. Intriguingly, emerging studies have revealed novel functions of Ephs in synapse formation and synaptic plasticity.⁵ Specific Ephs expressed in dendritic spines of adult brain are implicated in regulating spine morphogenesis, i.e., EphBs promote spine formation and maturation, while EphA4 induces spine retraction.^6,7In the adult hippocampus, EphA4 is localized to the dendritic spines.^7,8 Activation of EphA4 at the astrocyte-neuron contacts, triggered by astrocytic ephrin-A3, leads to spine retraction and results in a reduction of spine density.⁷ It has been well established that actin cytoskeletal rearrangement is critical for spine morphogenesis, and is controlled by a tight regulation of Rho GTPases including Rac1/Cdc42 and RhoA. Antagonistic regulation of Rac1/Cdc42 and RhoA has been observed to precede changes in spine morphogenesis, i.e., activation of Rac1/Cdc42 and inhibition of RhoA is involved in spine formation, and vice versa in spine retraction.⁹ Rho GTPases function as molecular switches that cycle between an inactive GDP-bound state and an active GTP-bound state. The activation status of GTPase is regulated by an antagonistic action of guanine-nucleotide exchange factors (GEFs) which enhance the exchange of bound GDP for GTP, and GTPase-activating proteins (GAPs) which increase the intrinsic rate of hydrolysis of bound GTP.¹⁰ Previous studies have implicated that Rho GTPases provides a direct link between Eph and actin cytoskeleton in diverse cellular processes including spine morphogenesis.¹¹ In particular, EphBs regulate spine morphology by modulating the activity of Rho GTPases, thereby leading to rearrangement of actin networks.^12–14 Although EphA4 activation results in spine shrinkage, the molecular mechanisms that underlie the action of EphA4 at dendritic spines remain largely unclear.Work from our laboratory recently demonstrated a critical role of cyclin-dependent kinase 5 (Cdk5) in mediating the action of EphA4 in spine morphogenesis through regulation of RhoA GTPase.¹⁵ Cdk5 is a proline-directed serine/threonine kinase initially identified to be a key regulator of neuronal differentiation, and has been implicated in actin dynamics through regulating the activity of Pak1, a Rac effector, during growth cone collapse and neurite outgrowth.¹⁶ We found that EphA4 stimulation by ephrin-A ligand enhances Cdk5 activity through phosphorylation of Cdk5 at Tyr15. More importantly, we demonstrated that ephexin1, a Rho GEF, is phosphorylated by Cdk5 in vivo. Ephexin1 was reported to transduce signals from activated EphA4 to RhoA, resulting in growth cone collapse during axon guidance.^17,18 Interestingly, we found that ephexin1 is highly expressed at the post-synaptic densities (PSDs) of adult brains.¹⁵ Loss of ephexin1 in cultured hippocampal neurons or in vivo perturbs the ability of ephrin-A to induce EphA4-dependent spine retraction. The loss of ephexin1 function in spine morphology can be rescued by reexpression of wild-type ephexin1, but not by expression of its phosphorylation-deficient mutant. Our findings therefore provide important evidence that phosphorylation of ephexin1 by Cdk5 is required for the EphA4-dependent spine retraction.Molecular mechanisms underlying the action of Cdk5/ephexin1 on actin networks in EphA4-mediated spine retraction is just beginning to be unraveled. It was reported that activation of EphA4-signaling induces tyrosine phosphorylation of ephexin1 through Src family kinases (SFKs), and promotes its exchange activity towards RhoA.¹⁷ Interestingly, mutation of the Cdk5 phosphorylation sites of ephexin1 attenuates the Src-dependent tyrosine phosphorylation of ephexin1 at Tyr⁸⁷ upon EphA4 activation. These findings suggest that Cdk5 is the “priming” kinase for ephexin1. We propose that EphA4 activation by ephrin-A ligand increases Cdk5 activity, leading to phosphorylation and priming of ephexin1 for the subsequent phosphorylation of ephexin1 by Src kinase at Tyr⁸⁷, resulting in an increase of its exchange activity towards RhoA. Thus, regulation of Cdk5 activity might indirectly control the phosphorylation of ephexin1 by Src. It is tempting to speculate that phosphorylation of ephexin1 by Cdk5 at the amino-terminal region leads to a conformational change of protein, thus facilitating the access of Tyr⁸⁷ site on ephexin1 to Src kinase. Whereas accumulating evidence have pointed to a pivotal role of various GEFs including Tiam1, intersectin and kalirin in regulating spine morphogenesis, the involvement of GAPs is not clear. For example, oligophrenin-1, a Rho GAP, is implicated in maintaining the spine length through repressing RhoA activity.¹⁹ Thus, it is conceivable that a specific GAP is involved in EphA4-dependent spine retraction. Recently, we found that α2-chimaerin, a Rac GAP, regulates EphA4-dependent signaling in hippocampal neurons (Shi and Ip, unpublished observations). Taken into consideration that α2-chimaerin is enriched in the PSDs, α2-chimaerin is a likely candidate that cooperates with ephexin1 during EphA4-dependent spine retraction.In addition to stimulation of the RTK signaling cascade following EphA4 receptor activation, clustering of EphA4 signaling complex is required for eliciting maximal EphA4 function.²⁰ It is tempting to speculate that Cdk5 also regulates the formation of EphA4-containing clusters in neurons. Indeed, Cdk5^-/- neurons show reduced size of EphA4 clusters upon ephrin-A treatment, suggesting that Cdk5 regulates the recruitment of downstream signaling proteins to activate EphA4. Moreover, since ephrinA-EphA4 interaction stimulates the activity of Cdk5 at synaptic contacts, it is possible that Cdk5 might play additional roles at the post-synaptic regions through phosphorylation of its substrates. For example, PSD-95, the major scaffold protein in the PSDs, and NMDA receptor subunit NR2A are both substrates for Cdk5. Interestingly, phosphorylation of these proteins by Cdk5 has been implicated in regulating the clustering of neurotransmitter receptors as well as synaptic transmission.^21,22 Consistent with these observations, spatial distribution of neurotransmitter receptors at neuromuscular synapses is altered and abnormal neurotransmission is observed in Cdk5^-/- mice.²³ Thus, further analysis to delineate the precise roles of Cdk5 in EphA4-dependent synapse development, including regulation of neurotransmitter receptor clustering, is required.Recently, Cdk5 was shown to regulate dendritic spine density and shape through controlling the phosphorylation status of Wiskott-Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE-1), a critical component of actin cytoskeletal network.²⁴ In particular, phosphorylation of WAVE-1 by Cdk5 prevents actin from Arp2/3 complex-dependent polymerization and leads to a loss of dendritic spines at basal state, while reduced Cdk5-dependent phosphorylation of WAVE-1 through cAMP-dependent dephosphorylation leads to an enhanced actin polymerization and increased number of spines. It is interesting to note that phosphorylation of ephexin1 and WAVE-1 by Cdk5 both results in a reduction of spine density. Whether a concerted phosphorylation of these proteins at synapses by Cdk5 plays a role in synaptic plasticity awaits further studies. Precise regulation of Cdk5 activity is unequivocally important to maintain its proper functions at synaptic contacts. Activation of Cdk5 is mainly dependent on its binding to two neuronal-specific activators, p35 or p39, and its activity can be enhanced upon phosphorylation at Tyr¹⁵.While the signals that lie upstream of Cdk5 have barely begun to be unraveled, Cdk5 has been demonstrated to be a key downstream regulator of signaling pathways activated by extracellular cues such as neuregulin, BDNF and semaphorin. To the best of our knowledge, ephrin-EphA4 signaling is the first extracellular cue that has been identified to phosphorylate Cdk5 and promote its activity at CNS synapses.^15,25 Since BDNF-TrkB and semaphorin3A-fyn signaling have also been implicated in synapse/ spine development, it is of importance to examine whether Cdk5 is the downstream integrator of these signaling events at synapses during spine morphogenesis.^26,27Although accumulating evidence highlights a role of Cdk5 in spatial learning and synaptic plasticity, the molecular mechanisms underlying the action of Cdk5 are largely unclear.^28,29 With the recent findings that reveal the critical involvement of Cdk5 in the regulation of Rho GTPases to affect spine morphology, it can be anticipated that precise regulation of actin dynamics by Cdk5 at synapses will be an important mechanism underlying synaptic plasticity in the adult brain.? Open in a separate windowFigure 1Phosphorylation of actin regulators by Cdk5 during dendritic spine morphogenesis. (A) In striatal and hippocampal neurons, phosphorylation of WAVE-1 by Cdk5 at basal condition prevents WAVE-1-mediated actin polymerization and leads to a loss of dendritic spines. However, activation of cyclic AMP-dependent signaling by neurotransmitter such as dopamine, reduces the Cdk5-dependent phosphorylation of WAVE-1 in these neurons. Dephosphorylation of WAVE-1 promotes actin polymerization and results in an increased number of mature dendritic spines. (B) In mature hippocampal neurons, activation of EphA4 by ephrin-A increases Cdk5-dependent of ephexin1. The phosphorylation of ephexin1 by Cdk5 facilitates its EphA4-stimulated GEF activity towards RhoA activation and leads to spine retraction.     

Hypothalamic NPY,AGRP, and POMC mRNA responses to leptin and refeeding in mice

Food deprivation (FD) increases hypothalamic neuropeptide Y (NPY) and agouti-related protein (AGRP) mRNA levels and decreases proopiomelanocortin (POMC) mRNA levels; refeeding restores these levels. We determined the time course of changes in hypothalamic NPY, AGRP, and POMC mRNA levels on refeeding after 24 h FD in C57BL mice by in situ hybridization. After 24 h deprivation, mice were refed with either chow or a palatable mash containing no calories or were injected with murine leptin (100 microg) without food. Mice were perfused 2 or 6 h after treatment. Food deprivation increased hypothalamic NPY mRNA (108 +/- 6%) and AGRP mRNA (78 +/- 7%) and decreased hypothalamic POMC mRNA (-15 +/- 1%). Refeeding for 6 h, but not 2 h, was sufficient to reduce (but not restore) NPY mRNA, did not affect AGRP mRNA, and restored POMC mRNA levels to ad libitum control levels. Intake of the noncaloric mash had no effect on mRNA levels, and leptin administration after deprivation (at a dose sufficient to reduce refeeding in FD mice) was not sufficient to affect mRNA levels. These results suggest that gradual postabsorptive events subsequent to refeeding are required for the restoration of peptide mRNA to baseline levels after food deprivation in mice.     

Anammox bacteria enrichment in upflow anaerobic sludge blanket (UASB) reactor

We investigated the anaerobic ammonium oxidation (anammox) reaction in a labscale upflow anaerobic sludge blanket (UASB) reactor. Our aim was to detect and enrich the organisms responsible for the anammox reaction using a synthetic medium that contained low concentrations of substrates (ammonium and nitrite). The reactor was inoculated with granular sludge collected from a full-scale anaerobic digestor used for treating brewery wastewater. The experiment was performed during 260 days under conditions of constant ammonium concentration (50 mg NH₄/⁺-N/L) and different nitrite concentrations (50∼150 mg NO₂-N/L). After 200 days, anammox activity was observed in the system. The microorganisms involved in this anammox reaction were identified as CandidatusB. Anammoxidans andK. Stuttgartiensis using fluorescencein situ hybridization (FISH) method.     

Interspecific variation of body shape and sexual dimorphism in three coexisting species of the genus <Emphasis Type="Italic">Petrotilapia</Emphasis> (Teleostei: Cichlidae) from Lake Malawi

Differences in color patterns have been the most used feature in describing cichlid species belonging to genus Petrotilapia from Lake Malawi. In this study, we quantified morphological variation in body shape within and among three coexisting Petrotilapia species using landmark-based geometric morphometric methods. Statistic analyses revealed significant body shape differences among species but not between sexes. Post hoc multiple comparisons based on Mahalanobis distances revealed that P. nigra was significantly different from P. genalutea and Petrotilapia sp., whereas the latter two were not significantly different. The splines generated showed that the most pronounced variation was in the head region, in which P. nigra had a relatively longer and deeper head than the other two. The most clear-cut distinction was in gape length; P. genalutea had the longest gape, followed by Petrotilapia sp., whereas P. nigra had the shortest gape. Body depth was shallower in P. nigra than the others. When comparing sexes by their centroid size, ANOVA revealed that males were bigger than females. Therefore, we conclude that color is not the only feature that can distinguish these congeners. We discuss the observed sexual dimorphism in terms of sexual selection and relate morphological variation among species to feeding behavior, which may help explain their coexistence in nature.     

Early biosignature of oxidative stress in the retinal pigment epithelium

The retinal pigment epithelium (RPE) is essential for retinoid recycling and phagocytosis of photoreceptors. Understanding of proteome changes that mediate oxidative stress-induced degeneration of RPE cells may provide further insight into the molecular mechanisms of retinal diseases. In the current study, comparative proteomics has been applied to investigate global changes of RPE proteins under oxidative stress. Proteomic techniques, including 2D SDS-PAGE, differential gel electrophoresis (DIGE), and tandem time-of-flight (TOF-TOF) mass spectrometry, were used to identify early protein markers of oxidative stress in the RPE. Two biological models of RPE cells revealed several differentially expressed proteins that are involved in key cellular processes such as energy metabolism, protein folding, redox homeostasis, cell differentiation, and retinoid metabolism. Our results provide a new perspective on early signaling molecules of redox imbalance in the RPE and putative therapeutic target proteins of RPE diseases caused by oxidative stress.     

Enhanced butanol production in Clostridium acetobutylicum ATCC 824 by double overexpression of 6-phosphofructokinase and pyruvate kinase genes

This study elucidated the importance of two critical enzymes in the regulation of butanol production in Clostridium acetobutylicum ATCC 824. Overexpression of both the 6-phosphofructokinase (pfkA) and pyruvate kinase (pykA) genes increased intracellular concentrations of ATP and NADH and also resistance to butanol toxicity. Marked increases of butanol and ethanol production, but not acetone, were also observed in batch fermentation. The butanol and ethanol concentrations were 29.4 and 85.5 % higher, respectively, in the fermentation by double-overexpressed C. acetobutylicum ATCC 824/pfkA+pykA than the wild-type strain. Furthermore, when fed-batch fermentation using glucose was carried out, the butanol and total solvent (acetone, butanol, and ethanol) concentrations reached as high as 19.12 and 28.02 g/L, respectively. The reason for improved butanol formation was attributed to the enhanced NADH and ATP concentrations and increased tolerance to butanol in the double-overexpressed strain.