Disrupted Ultradian Activity Rhythms and Differential Expression of Several Clock Genes in Interleukin-6-Deficient Mice.

The characteristics of the cycles of activity and rest stand out among the most intensively investigated aspects of circadian rhythmicity in humans and experimental animals. Alterations in the circadian patterns of activity and rest are strongly linked to cognitive and emotional dysfunctions in severe mental illnesses such as Alzheimer's disease (AD) and major depression (MDD). The proinflammatory cytokine interleukin 6 (IL-6) has been prominently associated with the pathogenesis of AD and MDD. However, the potential involvement of IL-6 in the modulation of the diurnal rhythms of activity and rest has not been investigated. Here, we set out to study the role of IL-6 in circadian rhythmicity through the characterization of patterns of behavioral locomotor activity in IL-6 knockout (IL-6 KO) mice and wild-type littermate controls. Deletion of IL-6 did not alter the length of the circadian period or the amount of locomotor activity under either light-entrained or free-running conditions. IL-6 KO mice also presented a normal phase shift in response to light exposure at night. However, the temporal architecture of the behavioral rhythmicity throughout the day, as characterized by the quantity of ultradian activity bouts, was significantly impaired under light-entrained and free-running conditions in IL-6 KO. Moreover, the assessment of clock gene expression in the hippocampus, a brain region involved in AD and depression, revealed altered levels of cry1, dec2, and rev-erb-beta in IL-6 KO mice. These data propose that IL-6 participates in the regulation of ultradian activity/rest rhythmicity and clock gene expression in the mammalian brain. Furthermore, we propose IL-6-dependent circadian misalignment as a common pathogenetic principle in some neurodegenerative and neuropsychiatric disorders.

Anhedonic behavior in cryptochrome 2-deficient mice is paralleled by altered diurnal patterns of amygdala gene expression.

Mood disorders are frequently paralleled by disturbances in circadian rhythm-related physiological and behavioral states and genetic variants of clock genes have been associated with depression. Cryptochrome 2 (Cry2) is one of the core components of the molecular circadian machinery which has been linked to depression, both, in patients suffering from the disease and animal models of the disorder. Despite this circumstantial evidence, a direct causal relationship between Cry2 expression and depression has not been established. Here, a genetic mouse model of Cry2 deficiency (Cry2 (-/-) mice) was employed to test the direct relevance of Cry2 for depression-like behavior. Augmented anhedonic behavior in the sucrose preference test, without alterations in behavioral despair, was observed in Cry2 (-/-) mice. The novelty suppressed feeding paradigm revealed reduced hyponeophagia in Cry2 (-/-) mice compared to wild-type littermates. Given the importance of the amygdala in the regulation of emotion and their relevance for the pathophysiology of depression, potential alterations in diurnal patterns of basolateral amygdala gene expression in Cry2 (-/-) mice were investigated focusing on core clock genes and neurotrophic factor systems implicated in the pathophysiology of depression. Differential expression of the clock gene Bhlhe40 and the neurotrophic factor Vegfb were found in the beginning of the active (dark) phase in Cry2 (-/-) compared to wild-type animals. Furthermore, amygdala tissue of Cry2 (-/-) mice contained lower levels of Bdnf-III. Collectively, these results indicate that Cry2 exerts a critical role in the control of depression-related emotional states and modulates the chronobiological gene expression profile in the mouse amygdala.

STAT3 controls IL6-dependent regulation of serotonin transporter function and depression-like behavior.

Experimental evidence suggests a role for the immune system in the pathophysiology of depression. A specific involvement of the proinflammatory cytokine interleukin 6 (IL6) in both, patients suffering from the disease and pertinent animal models, has been proposed. However, it is not clear how IL6 impinges on neurotransmission and thus contributes to depression. Here we tested the hypothesis that IL6-induced modulation of serotonergic neurotransmission through the STAT3 signaling pathway contributes to the role of IL6 in depression. Addition of IL6 to JAR cells, endogenously expressing SERT, reduced SERT activity and downregulated SERT mRNA and protein levels. Similarly, SERT expression was reduced upon IL6 treatment in the mouse hippocampus. Conversely, hippocampal tissue of IL6-KO mice contained elevated levels of SERT and IL6-KO mice displayed a reduction in depression-like behavior and blunted response to acute antidepressant treatment. STAT3 IL6-dependently associated with the SERT promoter and inhibition of STAT3 blocked the effect of IL6 in-vitro and modulated depression-like behavior in-vivo. These observations demonstrate that IL6 directly controls SERT levels and consequently serotonin reuptake and identify STAT3-dependent regulation of SERT as conceivable neurobiological substrate for the involvement of IL6 in depression.

Diurnal oscillation of amygdala clock gene expression and loss of synchrony in a mouse model of depression.

BACKGROUND: Disturbances in circadian rhythm-related physiological and behavioral processes are frequently observed in depressed patients and several clock genes have been identified as risk factors for the development of mood disorders. However, the particular involvement of the circadian system in the pathophysiology of depression and its molecular regulatory interface is incompletely understood. METHODS: A naturalistic animal model of depression based upon exposure to chronic mild stress was used to induce anhedonic behavior in mice. Micro-punch dissection was used to isolate basolateral amygdala tissue from anhedonic mice followed by quantitative real-time PCR-based analysis of gene expression. RESULTS: Here we demonstrate that chronic mild stress-induced anhedonic behavior is associated with disturbed diurnal oscillation of the expression of Clock, Cry2, Per1, Per3, Id2, Rev-erbα, Ror-ß and Ror-γ in the mouse basolateral amygdala. Clock gene desynchronization was accompanied by disruption of the diurnal expressional pattern of vascular endothelial growth factor A expression in the basolateral amygdala of anhedonic mice, also reflected in alterations of circulating vascular endothelial growth factor A levels. CONCLUSION: We propose that aberrant control of diurnal rhythmicity related to depression may indeed directly result from the illness itself and establish an animal model for the further exploration of the molecular mechanisms mediating the involvement of the circadian system in the pathophysiology of mood disorders.

Circadian abnormalities in a mouse model of high trait anxiety and depression.

INTRODUCTION: Dysregulation of circadian rhythms is a key symptom of mood disorders, including anxiety disorders and depression. Whether the circadian abnormalities observed in depressed patients are cause or consequence of the disease remains elusive. Here we aimed to explore potential disturbances of circadian rhythms in a validated genetic animal model of high trait anxiety and co-morbid depression and examine its molecular correlates. MATERIALS AND METHODS: Mice selectively bred for high (HAB) and normal (NAB) anxiety- and co-segregating depression-like behavior were subjected to analysis of circadian wheel-running activity to determine light-entrained (LD) and free-running circadian (DD) rhythms and a light-induced phase shift. Clock gene expression in HAB/NAB hippocampal tissue was analyzed by qRT-PCR and verified by Western blotting. RESULTS: Compared to NABs, HAB mice were found to present with altered DD length of daily cycle, fragmented ultradiem rhythms, and a blunted phase shift response. Clock gene expression analysis revealed a selective reduction of Cry2 expression in hippocampal tissue of HAB mice. DISCUSSION: We provide first evidence for a dysregulation of circadian rhythms in a mouse model of anxiety and co-morbid depression which suggests an association between depression and altered circadian rhythms at the genetic level and points towards a role for Cry2.

The role of post-translational modifications for learning and memory formation.

Learning and memory depend on molecular mechanisms involving the protein machinery. Recent evidence proposes that post-translational modifications (PTMs) play a major role in these cognitive processes. PTMs including phosphorylation of serine, threonine, and tyrosine are already well-documented to play a role for synaptic plasticity of the brain, neurotransmitter release, vesicle trafficking and synaptosomal or synaptosomal-associated proteins are substrates of a series of specific protein kinases and their counterparts, protein phosphatases. But protein phosphorylation is only one out of many possible PTMs and first work shows a role of palmitoylation as well as glycosylation for proteins involved in memory formation. Recent technology may now allow reliable detection and even quantification of PTMs of proteins involved in the cognitive system. This will contribute to the understanding of mechanisms for learning and memory formation at the chemical level and has to complement determination of protein levels and indeed determination of protein expression per se generates limited information. The many other PTMs expected including protein nitrosylation and alkylation will even represent targets for pharmacological interventions but in turn increase the complexity of the system. Nevertheless, determination of the presence and the function of PTMs is mandatory and promising cognitive research at the protein chemical level.

Modulation of the hippocampal protein machinery in voluntary and treadmill exercising rats.

Information about protein expression studies in the brain of exercising and sedentary animals is limited. Cognitive functions change during exercise and the aim of this study was to investigate rat protein levels of the protein machinery in the hippocampus, the main cognitive brain area for spatial learning and memory, in exercising rats. Protein fluctuations may reflect functional variation during exercise. Male Sprague-Dawley rats, 23 months old, were used for the study: the first group consisted of sedentary rats, the second of rats undertaking voluntary exercise from 5 months to 23 months and the third undertaking involuntary exercise on a treadmill from 5 months to 23 months. Two-dimensional gel electrophoresis with subsequent mass spectrometrical identification assigning spots to proteins and determination of coomassie-densities was carried out. Heterogeneous nuclear ribonucleoprotein K, one protein variant of heat shock cognate 71 kDa protein and BAG family molecular chaperone regulator 5 showed differential protein levels in the three groups when a p-value of <0.005 was considered as statistically significant thus respecting multiple testing. The biological meaning of changed protein levels in hippocampus under different conditions of exercise is not known but warrants further investigation.

Cognitive enhancement by SGS742 in OF1 mice is linked to specific hippocampal protein expression.

Cognitive enhancement by the GABA (B) antagonist SGS742 has been reported and we decided to search for proteins involved. Hippocampi from OF1 mice were from SGS742- treated animals and three control groups. Proteins were extracted and run on 2DE, and spots were quantified. Significantly different protein spots were identified by two mass spectrometry principles, nano-LC-ESI-MS/MS and by nano-LC-ESI(CID/ETD)-MS/MS. Signaling, chaperone and metabolic enzyme proteins were linked to memory enhancement.

Fluctuations of hippocampal neuronal protein levels over the estrous cycle in the rat.

Hippocampal function is known to be estrous-cycle-dependent but information on estrous-cycle-dependent protein expression is limited. It was therefore the aim to study protein levels of the neuronal network over the estrous cycle in the hippocampus of female rats and in males showing protein chemical neuroanatomy in this area. Female and male OFA Sprague-Dawley rats were used and females were grouped to proestrous, estrous, metestrous and diestrous by using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Spot volumes were quantified with specific software. A Synapsin-1 expression form was differentially regulated between proestrous and diestrous, a Synapsin IIa expression form was differentially regulated between proestrous and metestrous, the sum of ERC-2 proteins organizing the cytomatrix at the nerve terminals active zone was showing sex-dependent levels in the proestrous phase and Neurofilament triplet L protein was differentially expressed between the estrous phase and males. The findings may represent estrous-cycle-dependent hippocampal synaptic function that has been shown already in terms of electrophysiology and neuroanatomy. Neurofilament changes over the estrous cycle may reflect endoskeleton changes over the estrous cycle. We learn from this study, although increasing complexity of protein knowledge, that the estrous cycle and not only the sex per se has to be taken into account for design of future studies and interpretation of previous work at the protein level.

Mass spectrometrical characterization of NDRG2 protein (N-myc-downstream regulated gene 2) and description of two novel phosphorylation sites.

Antidepressant-related protein (NDRG2) is a member of the N-myc downstream-regulated gene family and a role for differentiation and signaling has been proposed. Performing protein profiling we observed NDRG2 and decided to characterize this important biomolecule. Estrous cycle phases were determined in Sprague-Dawley rats and the hippocampus was taken. Proteins were extracted, run on two-dimensional gel electrophoresis with subsequent multi-enzyme digestion followed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS analysis of spots. Spots identified as NDRG2 were quantified by specific software. Five spots were identified as NDRG2 and two novel phosphorylation sites (T330 and T334) were detected. Gender and estrous cycle-dependent NDRG2 levels were observed. Results are of importance for further qualitative and quantitative studies at the protein level as well as for the design of antibodies for immunochemical applications and for the interpretation of previous studies on NDRG2 that did not take into account different expression forms and posttranslational modifications.

Hippocampal signaling protein levels are different in early and late metestrus in the rat.

Early and late metestrus in the rat differ by progesterone levels. As it is known that progesterone shows a potential negative effect on cognitive performances and can counteract the estradiol-induced neural effects, we intended to study signaling proteins in the hippocampus, a structure representing a main brain area of cognitive function. Female OFA Sprague-Dawley rats were used in the studies and estrous phases were determined using vaginal smears. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Individual signaling protein levels quantified by specific software were shown to vary between the two phases, including NG,NG-dimethylarginine dimethylaminohydrolase 1 for nitric oxide signaling, guanine nucleotide-binding proteins, septin 6, septin 11, G-septin alpha, and 14-3-3 protein gamma. Results from this study indicate that early and late metestrus show differences in signaling pathways, that may help to design further investigations at the protein level and may assist to interpret literature on protein expression and brain protein levels in female rats. Moreover, signaling differences in hippocampus are challenging cognitive studies during these two metestrus phases probably revealing cognitive differences between early and late metestrus.

Estrous-cycle-dependent hippocampal levels of signaling proteins.

There is information that proteins are expressed in a hormone-dependent manner but no systematic study on this subject has been carried out to the best of our knowledge. We therefore decided to investigate protein expression in a well-studied brain area, the hippocampus, in female rats at various phases of the estrous cycle and in male rats. Male and female OFA Sprague-Dawley rats were used in the studies and estrous phases were determined using vaginal smears and females were grouped according to PE, E, ME, and DE. Hippocampal tissue was taken, proteins extracted, run on two-dimensional gel electrophoresis and proteins were identified by mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Individual signaling protein levels quantified by specific software were shown to depend on sex and phase of the estrous cycle. These include NG,NG-dimethylarginine dimethylaminohydrolase for nitric oxide signaling, stathmin, SH3 domain protein 2A, SH3 domain protein 2B, S100 calcium binding protein B, calcyclin-binding protein, Syndapin I, GTPase HRas, guanine nucleotide-binding proteins, septin 8, G-septin alpha, phosphtidylethanolamine-binding protein, several protein phosphatases. Results from this study, although increasing complexity of protein knowledge, may help to design further investigations at the protein level and may assist to interpret literature on protein expression and brain protein levels.

The hippocampal protein machinery varies over the estrous cycle.

Information about estrous cycle (EC) and sex-dependent protein levels is limited. Cognitive functions vary over the EC and the aim of this study was to investigate rat protein fluctuations in the hippocampus, the main cognitive brain area for learning and memory, in the individual phases of the EC and in males and indeed protein fluctuations may reflect functional variation over the EC. Sprague-Dawley rats were used in the studies and estrous phases were determined. Hippocampi were taken, proteins extracted, run on 2-DE, and identified using MALDI-TOF/TOF and nano-LC-ESI-MS/MS; protein levels were quantified using Proteomweaver software. Levels of protein synthetic machinery components transcriptional activator protein PUR(α,ß), elongation factor 2, heterogeneous nuclear ribonucleoprotein K, chaperones 78 kDa glucose-regulated protein, heat shock cognate 71 kDa protein, Hsp 105, stress-70 protein, peptidyl-prolyl cis-trans isomerase A, prefoldin subunit 2, T-complex protein 1 subunit alpha and subunit delta, and degradation principle proteasome subunit alpha type 1 and ubiquitin carboxyl-terminal hydrolase isozyme L1, were different between sex and phase of the EC. We suggest that differences in the protein synthetic, chaperoning, and degradation machinery indicate different function in the individual EC phases. Results herein are relevant for further design of studies in the hippocampus at the protein level and interpretation of previous studies because EC phases will have to be respected and taken into account.

Serum, liver, and kidney proteomic analysis for the alloxan-induced type I diabetic mice after insulin gene transfer of naked plasmid through electroporation.

Gene therapy has been reported to be effective in treating diabetes mellitus (DM), while little has been found out about the functional protein changes since. The liver and kidney play important roles in glucose absorption, metabolism, and excretion. Changes in the two organs may reflect pathologic alterations during DM, while the serum has a direct connection with most organs and pathological changes. We used alloxan to induce diabetic mice, electrotranferred the insulin gene into their sural muscles, and discovered that their blood glucose decreased to normal level. Consequently, proteomic approaches were applied to evaluate protein changes in the liver, kidney, and serum of normal, diabetic, and gene transferred mice. Forty-three proteins were found either up-regulated or down-reglulated in the liver, kidney, and serum of the alloxan-induced type I diabetic mice. Only five proteins in the liver, five proteins in the kidney, and seven proteins in the serum of diabetic mice were found to be back-regulated to normal levels after gene transfer. These back-regulated proteins are involved in lipid and glucose metabolism, associated with phosphorylation, signal transduction, oxidation, and immune inflammation. Our findings might promote a better understanding for the mechanism of DM, and provide novel targets for estimating the effects of gene therapy.

Bone loss induced by ovariectomy in rats is prevented by gene transfer of parathyroid hormone or an Arg-Gly-Asp-containing peptide.

Osteoporosis is a major and growing healthcare concern as the population ages. The genes of both a Arg-Gly-Asp (RGD)-containing peptide and parathyroid hormone (PTH) were used to reduce bone loss induced by ovariectomy (OVX) in rats. Plasmids with either RGD or PTH gene were delivered into the quadriceps of OVX rats. The expression of the genes was detected by RT-PCR and radioimmunoassay. Analysis of bone mineral density, bone mechanical testing and bone mineral content indicated an improvement in bone properties in both RGD-transferred and PTH-transferred rats compared to OVX rats. Gene transfer of either RGD or PTH is therefore a possible approach to prevent bone loss in OVX rats thus providing a potential method to prevent osteoporosis in clinical situations.

Transfer of a gene containing the Arg-Gly-Asp peptide prolongs the bleeding time of mice.

Peptides containing Arg-Gly-Asp (RGD) have been used to decrease thrombosis by competitive inhibition of the integrin glycoprotein, alphaIIb/beta3a, in platelets. However, they have a short half-life in vivo. A naked plasmid, pCMV-RGD, was transferred into the skeletal muscle of mice and RGD gene expression was observed by RT-PCR. The bleeding time between control mice and RGD-transferred mice was prolonged from the 10th day to the 80th day after gene transfer while the blood glucose and serum insulin-like proteins remained at normal levels. These results provided a convenient and effective approach to relieve patients from thrombi in a single step over a relatively long period.