Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.

Diet-induced obesity alters the differentiation potential of stem cells isolated from bone marrow, adipose tissue and infrapatellar fat pad: the effects of free fatty acids.

INTRODUCTION: Obesity is a major risk factor for several musculoskeletal conditions that are characterized by an imbalance of tissue remodeling. Adult stem cells are closely associated with the remodeling and potential repair of several mesodermally derived tissues such as fat, bone and cartilage. We hypothesized that obesity would alter the frequency, proliferation, multipotency and immunophenotype of adult stem cells from a variety of tissues. MATERIALS AND METHODS: Bone marrow-derived mesenchymal stem cells (MSCs), subcutaneous adipose-derived stem cells (sqASCs) and infrapatellar fat pad-derived stem cells (IFP cells) were isolated from lean and high-fat diet-induced obese mice, and their cellular properties were examined. To test the hypothesis that changes in stem cell properties were due to the increased systemic levels of free fatty acids (FFAs), we further investigated the effects of FFAs on lean stem cells in vitro. RESULTS: Obese mice showed a trend toward increased prevalence of MSCs and sqASCs in the stromal tissues. While no significant differences in cell proliferation were observed in vitro, the differentiation potential of all types of stem cells was altered by obesity. MSCs from obese mice demonstrated decreased adipogenic, osteogenic and chondrogenic potential. Obese sqASCs and IFP cells showed increased adipogenic and osteogenic differentiation, but decreased chondrogenic ability. Obese MSCs also showed decreased CD105 and increased platelet-derived growth factor receptor α expression, consistent with decreased chondrogenic potential. FFA treatment of lean stem cells significantly altered their multipotency but did not completely recapitulate the properties of obese stem cells. CONCLUSIONS: These findings support the hypothesis that obesity alters the properties of adult stem cells in a manner that depends on the cell source. These effects may be regulated in part by increased levels of FFAs, but may involve other obesity-associated cytokines. These findings contribute to our understanding of mesenchymal tissue remodeling with obesity, as well as the development of autologous stem cell therapies for obese patients.

Ets-1 p27: A novel Ets-1 isoform with dominant-negative effects on the transcriptional properties and the subcellular localization of Ets-1 p51

The transcription factor Ets-1 is implicated in various physiological processes and invasive pathologies. We identified a novel variant of ets-1, ets-1Delta(III-VI), resulting from the alternative splicing of exons III to VI. This variant encodes a 27 kDa isoform, named Ets-1 p27. Ets-1 p27 lacks the threonine-38 residue, the Pointed domain and the transactivation domain, all of which are required for the transactivation of Ets-1 target genes. Both inhibitory domains surrounding the DNA-binding domain are conserved, suggesting that Ets-1 p27, like the full-length Ets-1 p51 isoform, is autoinhibited for DNA binding. We showed that Ets-1 p27 binds DNA in the same way as Ets-1 p51 does and that it acts both at a transcriptional and a subcellular localization level, thereby constituting a dual-acting dominant negative of Ets-1 p51. Ets-1 p27 blocks Ets-1 p51-mediated transactivation of target genes and induces the translocation of Ets-1 p51 from the nucleus to the cytoplasm. Furthermore, Ets-1 p27 overexpression represses the tumor properties of MDA-MB-231 mammary carcinoma cells in correlation with the known implication of Ets-1 in various cellular mechanisms. Thus the dual-acting dominant-negative function of Ets-1 p27 gives to the Ets-1 p27/Ets-1 p51 ratio a determining effect on cell fate.

Physiological levels of PTEN control the size of the cellular Ins(1,3,4,5,6)P5 pool

To understand how a signaling molecule's activities are regulated, we need insight into the processes controlling the dynamic balance between its synthesis and degradation. For the Ins(1,3,4,5,6)P5 signal, this information is woefully inadequate. For example, the only known cytosolic enzyme with the capacity to degrade Ins(1,3,4,5,6)P5 is the tumour-suppressor PTEN [J.J. Caffrey, T. Darden, M.R. Wenk, S.B. Shears, FEBS Lett. 499 (2001) 6 ], but the biological relevance has been questioned by others [E.A. Orchiston, D. Bennett, N.R. Leslie, R.G. Clarke, L. Winward, C.P. Downes, S.T. Safrany, J. Biol. Chem. 279 (2004) 1116 ]. The current study emphasizes the role of physiological levels of PTEN in Ins(1,3,4,5,6)P5 homeostasis. We employed two cell models. First, we used a human U87MG glioblastoma PTEN-null cell line that hosts an ecdysone-inducible PTEN expression system. Second, the human H1299 bronchial cell line, in which PTEN is hypomorphic due to promoter methylation, has been stably transfected with physiologically relevant levels of PTEN. In both models, a novel consequence of PTEN expression was to increase Ins(1,3,4,5,6)P5 pool size by 30-40% (p<0.01); this response was wortmannin-insensitive and, therefore, independent of the PtdIns 3-kinase pathway. In U87MG cells, induction of the G129R catalytically inactive PTEN mutant did not affect Ins(1,3,4,5,6)P(5) levels. PTEN induction did not alter the expression of enzymes participating in Ins(1,3,4,5,6)P5 synthesis. Another effect of PTEN expression in U87MG cells was to decrease InsP6 levels by 13% (p<0.02). The InsP6-phosphatase, MIPP, may be responsible for the latter effect; we show that recombinant human MIPP dephosphorylates InsP6 to D/L-Ins(1,2,4,5,6)P5, levels of which increased 60% (p<0.05) following PTEN expression in U87MG cells. Overall, our data add higher inositol phosphates to the list of important cellular regulators [Y. Huang, R.P. Wernyj, D.D. Norton, P. Precht, M.C. Seminario, R.L. Wange, Oncogene, 24 (2005) 3819 ] the levels of which are modulated by expression of the highly pleiotropic PTEN protein.

Bucephaloides-Gracilescens - A Quantitative Study Of The Lysosomal Cellular Stress Response Induced By The Sporocysts And Developing Cercariae Within The White Furrow Shell Abra-Alba

Reproductive stress is apparent inAbra alba as a result of infection with the sporocysts ofBucephaloides gracilescens, culminating in castration in heavily infected specimens. The bivalve is also subject to mechanical stress from actively growing sporocyst tubules and nutritional stress due to the nutrient requirement of large numbers of germ balls within the sporocysts. Using the digestive cell lysosomal system ofAbra as a monitor, it was possible to demonstrate quantitatively a parasite-induced cellular stress response by applying a sensitive cytochemical test for lysosomal stability. Lysosomal stability was determined as the labilisation period for latent Nacetyl-β-hexosaminidase (NAH), measured by microdensitometry. In uninfectedAbra, digestive cell lysosomal NAH expressed structure-linked latency. Hence a significantly longer labilisation period was required compared with infectedAbra, where the parasitic burden with its associated stress effects resulted in a destabilisation of the lysosomal membrane. This reduced the latency of the enzyme, so that a much shorter labilisation period was required for the stressed tissue to express maximum lysosomal enzyme activity. It is suggested that the lysosomal system of the digestive cells inAbra can be used as a sensitive monitor of the stress induced by the sporocysts and developing cercariae ofBucephaloides.

Some Practical Considerations In The Measurement Of Pollution Effects On Bivalve Mollusks And Some Possible Ecological Consequences

A consideration of some physiological (rates of oxygen consumption, the scope for growth) and cellular (the cytochemical latency of a lysosomal enzyme) processes in bivalve molluscs suggests that animal size and seasonal changes related to the gametogenic cycle are important sources of natural variability. Correcting for size using regression techniques, and limiting measurements to one part of the gametogenic cycle, reduces observed natural variability considerably. Differences between populations are then still apparent, but the results of laboratory experiments with hydrocarbons from crude oil suggest that it should be possible to detect sub-lethal effects due to pollution (the ‘signal’) in the presence of the remaining natural variability (the ‘noise’). Statistical considerations, taken together with results from current studies on Mytilus edulis and Scobicularia plana, indicate that sample sizes of 10–15 individuals should suffice for the detection of possible pollution effects. The physiological effects to be expected in the presence of sub-lethal levels of polluting hydrocarbons are on a scaie that can cause significant ecological damage to a population through a reduction in fecundity and the residual reproductive value of the individuals.

Interaction Effects of Light, Temperature and Nutrient Limitations (N, P and Si) on Growth, Stoichiometry and Photosynthetic Parameters of the Cold-Water Diatom Chaetoceros wighamii

Light (20-450 μmol photons m^-2 s^-1), temperature (3-11°C) and inorganic nutrient composition (nutrient replete and N, P and Si limitation) were manipulated to study their combined influence on growth, stoichiometry (C:N:P:Chl a) and primary production of the cold water diatom Chaetoceros wighamii. During exponential growth, the maximum growth rate (~0.8 d^-1) was observed at high temperture and light; at 3°C the growth rate was ~30% lower under similar light conditions. The interaction effect of light and temperature were clearly visible from growth and cellular stoichiometry. The average C:N:P molar ratio was 80:13:1 during exponential growth, but the range, due to different light acclimation, was widest at the lowest temperature, reaching very low C:P (~50) and N:P ratios (~8) at low light and temperature. The C:Chl a ratio had also a wider range at the lowest temperature during exponential growth, ranging 16-48 (weight ratio) at 3°C compared with 17-33 at 11°C. During exponential growth, there was no clear trend in the Chl a normalized, initial slope (α*) of the photosynthesis-irradiance (PE) curve, but the maximum photosynthetic production (P_m) was highest for cultures acclimated to the highest light and temperature. During the stationary growth phase, the stoichiometric relationship depended on the limiting nutrient, but with generally increasing C:N:P ratio. The average photosynthetic quotient (PQ) during exponential growth was 1.26 but decreased to <1 under nutrient and light limitation, probably due to photorespiration. The results clearly demonstrate that there are interaction effects between light, temperature and nutrient limitation, and the data suggests greater variability of key parameters at low temperature. Understanding these dynamics will be important for improving models of aquatic primary production and biogeochemical cycles in a warming climate.

Effects of roziglitazone and interactions with growth-regulating factors in ventricular cell hypertrophy

Chronic administration of thiazolidinediones might predispose to cardiac hypertrophy. The aim was to investigate direct effects of rosiglitazone in rat ventricular cardiomyocytes maintained in vitro (24 h). Rosiglitazone (=10-5 M) did not increase protein synthesis and produced small inconsistent increases in cellular protein. In the presence of serum (10% v/v), but not insulin-like growth factor (IGF-1, 10-8 M) or insulin (1 U/ml), an interaction with rosiglitazone to stimulate protein synthesis was observed. The hypertrophic responses to noradrenaline (5×10-6 M), PMA (10-7 M) and ET-1 (10-7 M) were not attenuated by rosiglitazone. Rosiglitazone (10-7 M) did not influence protein synthesis in response to insulin (1 U/ml) and elevated glucose (2.5×10-2 M) alone or in combination, but attenuated the increase in protein mass observed in response to elevated glucose alone. In re-differentiated cardiomyocytes, a model of established hypertrophy, rosiglitazone (10-8 M–10-6 M) increased protein synthesis. Together, these data indicate that rosiglitazone does not initiate cardiomyocyte hypertrophy directly in vitro. However, during chronic administration, the interaction of rosiglitazone with locally-derived growth-regulating factors may make a modest contribution to cardiac remodelling and influence the extent of compensatory hypertrophy of the compromised rat heart.

Differential effects of an anti-oxidant intervention on cardiomyocyte expression of adrenomedullin and intermedin and their receptor components in chronic nitric oxide deficiency

Background: Chronic inhibition of nitric oxide (NO) synthesis is associated with hypertension, myocardial oxidative stress and hypertrophic remodeling. Up-regulation of the cardiomyocyte adrenomedullin (AM) / intermedin (IMD) receptor signaling cascade is also apparent in NO-deficient cardiomyocytes: augmented expression of AM and receptor activity modifying proteins RAMP2 and RAMP3 is prevented by blood pressure normalization while that of RAMP1 and intermedin (IMD) is not, indicating that the latter is regulated by a pressure-independent mechanism. Aims: to verify the ability of an anti-oxidant intervention to normalize cardiomyocyte oxidant status and to investigate the influence of such an intervention on expression of AM, IMD and their receptor components in NO-deficient cardiomyocytes. Methods: NO synthesis inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 35mg/kg/day) was given to rats for 8 weeks, with/without con-current administration of antioxidants (Vitamin C (25mg/kg/day) and Tempol (25mg/kg/day)). Results: In left ventricular cardiomyocytes isolated from L-NAME treated rats, increased oxidative stress was indicated by augmented (3.6 fold) membrane protein oxidation, enhanced expression of catalytic and regulatory subunits of pro-oxidant NADPH oxidases (NOX1, NOX2) and compensatory increases in expression of anti-oxidant glutathione peroxidase and Cu/Zn superoxide dismutases (SOD1, SOD3). Vitamin C plus Tempol did not reduce systolic blood pressure but normalized augmented plasma levels of IMD, but not of AM, and in cardiomyocytes: (i) abolished increased membrane protein oxidation; (ii) normalized augmented expression of prepro-IMD and RAMP1, but not prepro-AM, RAMP2 and RAMP3; (iii) attenuated (by 42%) increased width and normalized expression of hypertrophic markers, skeletal-�-actin and prepro-endothelin-1 similarly to blood pressure normalization but in contrast to blood pressure normalization did not attenuate augmented brain natriuretic peptide (BNP) expression. Conclusion: normalization specifically of augmented IMD/RAMP1 expression in NO-deficient cardiomyocytes by antioxidant intervention in the absence of blood pressure reduction indicates that these genes are likely to be induced directly by myocardial oxidative stress. Although oxidative stress contributed to cardiomyocyte hypertrophy, induction of IMD and RAMP1 is unlikely to be secondary to cardiomyocyte hypertrophy.

Direct and indirect effects of obestatin peptides on food intake and the regulation of glucose homeostasis and insulin secretion in mice

Obestatin is a recently discovered peptide hormone that appears to be involved in reducing food intake, gut motility and body weight. Obestatin is a product of the preproghrelin gene and appears to oppose several physiological actions of ghrelin. This study investigated the acute effects of obestatin (1-23) and the truncated form, obestatin (11-23), on feeding activity, glucose homeostasis or insulin secretion. Mice received either intraperitoneal obestatin (1-23) or (11-23) (1 mu mol/kg) 4 h prior to an allowed 15 min period of feeding. Glucose excursions and insulin responses were lowered by 64-77% and 39-41%, respectively, compared with saline controls. However this was accompanied by 43% and 53% reductions in food intake, respectively. The effects of obestatin peptides were examined under either basal or glucose (18 mmol/kg) challenge conditions to establish whether effects were independent of changes in feeding. No alterations in plasma glucose or insulin responses were observed. In addition, obestatin peptides had no effect on insulin sensitivity as revealed by hypoglycaemic response when co-administered with insulin. Our observations support a role for obestatin in regulating metabolism through changes of appetite, but indicate no direct actions on glucose homeostasis or insulin secretion. (c) 2007 Elsevier Inc. All rights reserved.

Lys(9) for Glu(9) substitution in glucagon-like peptide-1(7-36)amide confers dipeptidylpeptidase IV resistance with cellular and metabolic actions similar to those of established antagonists glucagon-like peptide-1(9-36)amide and exendin (9-39)

The incretin hormone glucagon-like peptide-1(7-36)amide (GLP-1) has been deemed of considerable importance in the regulation of blood glucose. Its effects, mediated through the regulation of insulin, glucagon, and somatostatin, are glucose-dependent and contribute to the tight control of glucose levels. Much enthusiasm has been assigned to a possible role of GLP-1 in the treatment of type 2 diabetes. GLIP-l's action unfortunately is limited through enzymatic inactivation caused by dipeptidylpeptidase IV (DPP IV). It is now well established that modifying GLP-1 at the N-terminal amino acids, His(7) and Ala(8), can greatly improve resistance to this enzyme. Little research has assessed what effect Glu(9)-substitution has on GLP-1 activity and its degradation by DPP IV. Here, we report that the replacement of Glu(9) of GLP-1 with Lys dramatically increased resistance to DPP IV. This analogue, (Lys(9))GLP-1, exhibited a preserved GLP-1 receptor affinity, but the usual stimulatory effects of GLP-1 were completely eliminated, a trait duplicated by the other established GLP-1-antagonists, exendin (9-39) and GLP-1 (9-36)amide. We investigated the in vivo antagonistic actions of (Lys(9))GLP-1 in comparison with GLP-1(9-36)amide and exendin (9-39) and revealed that this novel analogue may serve as a functional antagonist of the GLP-1 receptor. (C) 2004 Elsevier Inc. All rights reserved.