CCL17/thymus and activation-regulated chemokine induces calcitonin gene-related peptide in human airway epithelial cells through CCR4.

BACKGROUND: Calcitonin gene-related peptide (CGRP) is a potent arterial and venous vasodilator. Increased airway epithelial cell expression of CGRP, together with increased CCL17 expression, was previously observed in a model of provoked asthma in atopic human subjects. OBJECTIVE: We sought to determine whether CCL17 induces CCR4-dependent CGRP synthesis and secretion by human airway epithelial cells. METHODS: Human airway epithelial cell lines (BEAS-2B and A549) and human primary airway cells were cultured with CCL17 or various other cytokines, and CGRP expression was measured by using RT-PCR, quantitative immunofluorescence, and enzyme immunoassay. CCR4 expression was determined in cultured cells by using flow cytometry and in bronchial biopsy specimens by using immunohistochemistry. RESULTS: CCL17 induced a several thousand-fold increase in CGRP mRNA expression and released peptide product from BEAS-2B and A549 cells in a time- and concentration-dependent fashion. Concentration-dependent CCL17-induced release of CGRP by primary human airway epithelial cells was also observed. Under comparable conditions, CCL17 induced greater CGRP release from BEAS-2B cells than either IL-13, a cytokine mixture (TNF-α, GM-CSF, and IL-1), or CCL22. CCR4 was expressed by BEAS-2B and A549 cells and internalized after ligation with CCL17. CCL17-induced CGRP release was inhibited by a specific anti-CCR4 blocking antibody. Bronchial biopsy specimens obtained from healthy volunteers and asthmatic patients before and after provoked asthma all exhibited CCR4 staining of equivalent intensity, indicating that the receptor is constitutively expressed. CONCLUSIONS: CCL17-induced, CCR4-dependent release of CGRP by human airway epithelial cells represents a novel inflammatory pathway and a possible target in patients with asthma and allergic disease.

A mental retardation gene, motopsin/prss12, modulates cell morphology by interaction with seizure-related gene 6.

A serine protease, motopsin (prss12), plays a significant role in cognitive function and the development of the brain, since the loss of motopsin function causes severe mental retardation in humans and enhances social behavior in mice. Motopsin is activity-dependently secreted from neuronal cells, is captured around the synaptic cleft, and cleaves a proteoglycan, agrin. The multi-domain structure of motopsin, consisting of a signal peptide, a proline-rich domain, a kringle domain, three scavenger receptor cysteine-rich domains, and a protease domain at the C-terminal, suggests the interaction with other molecules through these domains. To identify a protein interacting with motopsin, we performed yeast two-hybrid screening and found that seizure-related gene 6 (sez-6), a transmembrane protein on the plasma membrane of neuronal cells, bound to the proline-rich/kringle domain of motopsin. Pull-down and immunoprecipitation analyses indicated the interaction between these proteins. Immunocytochemical and immunohistochemical analyses suggested the co-localization of motopsin and sez-6 at neuronal cells in the developmental mouse brain and at motor neurons in the anterior horn of human spinal cords. Transient expression of motopsin in neuro2a cells increased the number and length of neurites as well as the level of neurite branching. Interestingly, co-expression of sez-6 with motopsin restored the effect of motopsin at the basal level, while sez-6 expression alone showed no effects on cell morphology. Our results suggest that the interaction of motopsin and sez-6 modulates the neuronal cell morphology.

Chorein, the protein responsible for chorea-acanthocytosis, interacts with ß-adducin and ß-actin.

Chorea-acanthocytosis (ChAc) is an autosomal, recessive hereditary disease characterized by striatal neurodegeneration and acanthocytosis, and caused by loss of function mutations in the vacuolar protein sorting 13 homolog A (VPS13A) gene. VPS13A encodes chorein whose physiological function at the molecular level is poorly understood. In this study, we show that chorein interacts with ß-adducin and ß-actin. We first compare protein expression in human erythrocyte membranes using proteomic analysis. Protein levels of ß-adducin isoform 1 and ß-actin are markedly decreased in erythrocyte membranes from a ChAc patient. Subsequent co-immunoprecipitation (co-IP) and reverse co-IP assays using extracts from chorein-overexpressing human embryonic kidney 293 (HEK293) cells, shows that ß-adducin (isoforms 1 and 2) and ß-actin interact with chorein. Immunocytochemical analysis using chorein-overexpressing HEK293 cells demonstrates co-localization of chorein with ß-adducin and ß-actin. In addition, immunoreactivity of ß-adducin isoform 1 is significantly decreased in the striatum of gene-targeted ChAc-model mice. Adducin and actin are membrane cytoskeletal proteins, involved in synaptic function. Expression of ß-adducin is restricted to the brain and hematopoietic tissues, corresponding to the main pathological lesions of ChAc, and thereby implicating ß-adducin and ß-actin in ChAc pathogenesis.

Evaluation of the prostaglandin F synthase activity of human and bovine aldo-keto reductases: AKR1A1s complement AKR1B1s as potent PGF synthases.

AKR1B1 of the polyol pathway was identified as a prostaglandin F2α synthase (PGFS). Using a genomic approach we have identified in the endometrium five bovine and three human AKRs with putative PGFS activity and generated the corresponding recombinant enzymes. The PGFS activity of the recombinant proteins was evaluated using a novel assay based on in situ generation of the precursor of PG biosynthesis PGH2. PGF2α was measured by ELISA and the relative potencies of the different enzymes were compared. We identified AKR1A1 and confirmed AKR1B1 as the most potent PGFS expressing characteristic inhibition patterns in presence of methylglyoxal, ponalrestat and glucose.

Evaluation of the allergenicity of ω5-gliadin-deficient Hokushin wheat (1BS-18) in a wheat allergy rat model.

We previously developed Hokushin wheat line as a hypoallergenic wheat lacking ω5-gliadin (1BS-18), a major allergen for wheat-dependent exercise-induced anaphylaxis. However, the allergenicity of 1BS-18 has not been understood completely. In this study, we evaluated the allergenicity of 1BS-18 such as anaphylactic elicitation ability and sensitization ability using rats sensitized with ω5-gliadin or glutens prepared from Hokushin (Hokushin gluten) or 1BS-18 (1BS-18 gluten). Rats were sensitized by intraperitoneal administration of ω5-gliadin, Hokushin gluten or 1BS-18 gluten. Immunoglobulin E-mediated systemic anaphylaxis was evaluated by measuring changes in rectal temperature for 30 min after intravenous challenge with ω5-gliadin or the test glutens in unsensitized rats or rats sensitized with ω5-gliadin or the test glutens. In ω5-gliadin-sensitized rats, intravenous challenge with ω5-gliadin or Hokushin gluten significantly decreased the rectal temperature at 30 min after challenge while challenge with 1BS-18 gluten did not reduce the rectal temperature. Furthermore, intravenous challenge with ω5-gliadin significantly decreased the rectal temperature in rats sensitized with Hokushin gluten or 1BS-18 gluten. However, the reduced degree observed in 1BS-18 gluten-sensitized rats was smaller than that in Hokushin gluten-sensitized rats. In conclusion, 1BS-18 elicited no allergic reaction in ω5-gliadin-sensitized rats and had less sensitization ability for ω5-gliadin than that of Hokushin wheat.

Intravenous infusion of mesenchymal stem cells promotes functional recovery in a rat model of chronic cerebral infarction.

OBJECTIVE: Intravenous infusion of mesenchymal stem cells (MSCs) derived from adult bone marrow improves behavioral function in rat models of cerebral infarction. Although clinical studies are ongoing, most studies have focused on the acute or subacute phase of stroke. In the present study, MSCs derived from bone marrow of rats were intravenously infused 8 weeks after the induction of a middle cerebral artery occlusion (MCAO) to investigate whether delayed systemic injection of MSCs improves functional outcome in the chronic phase of stroke in rats. METHODS: Eight weeks after induction of the MCAO, the rats were randomized and intravenously infused with either MSCs or vehicle. Ischemic volume and behavioral performance were examined. Blood-brain barrier (BBB) integrity was assessed by quantifying the leakage of Evans blue into the brain parenchyma after intravenous infusion. Immunohistochemical analysis was also performed to evaluate the stability of the BBB. RESULTS: Motor recovery was better in the MSC-treated group than in the vehicle-treated group, with rapid improvement (evident at 1 week post-infusion). In MSC-treated rats, reduced BBB leakage and increased microvasculature/repair and neovascularization were observed. CONCLUSIONS: These results indicate that the systemic infusion of MSCs results in functional improvement, which is associated with structural changes in the chronic phase of cerebral infarction, including in the stabilization of the BBB.

Effects of early surgical decompression on functional and histological outcomes after severe experimental thoracic spinal cord injury.

OBJECTIVE In acute traumatic brain injury, decompressive craniectomy is a common treatment that involves the removal of bone from the cranium to relieve intracranial pressure. The present study investigated whether neurological function following a severe spinal cord injury improves after utilizing either a durotomy to decompress the intradural space and/or a duraplasty to maintain proper flow of cerebrospinal fluid. METHODS Sixty-four adult female rats (n = 64) were randomly assigned to receive either a 3- or 5-level decompressive laminectomy (Groups A and B), laminectomy + durotomy (Groups C and D), or laminectomy + duraplasty with graft (Group E and F) at 24 hours following a severe thoracic contusion injury (200 kilodynes). Duraplasty involved the use of DuraSeal, a hydrogel dural sealant. Uninjured and injured control groups were included (Groups G, H). Hindlimb locomotor function was assessed by open field locomotor testing (BBB) and CatWalk gait analysis at 35 days postinjury. Bladder function was analyzed and bladder wall thickness was assessed histologically. At 35 days postinjury, mechanical and thermal allodynia were assessed by the Von Frey hair filament and hotplate paw withdrawal tests, respectively. Thereafter, the spinal cords were dissected, examined for gross anomalies at the injury site, and harvested for histological analyses to assess lesion volumes and white matter sparing. ANOVA was used for statistical analyses. RESULTS There was no significant improvement in motor function recovery in any treatment groups compared with injured controls. CatWalk gait analysis indicated a significant decrease in interlimb coordination in Groups B, C, and D (p < 0.05) and swing speed in Groups A, B, and D. Increased mechanical pain sensitivity was observed in Groups A, C, and F (p < 0.05). Rats in Group C also developed thermal pain hypersensitivity. Examination of spinal cords demonstrated increased lesion volumes in Groups C and F and increased white matter sparing in Group E (p < 0.05). The return of bladder automaticity was similar in all groups. Examination of the injury site during tissue harvest revealed that, in some instances, expansion of the hydrogel dural sealant caused compression of the spinal cord. CONCLUSIONS Surgical decompression provided no benefit in terms of neurological improvement in the setting of a severe thoracic spinal cord contusion injury in rats at 24 hours postinjury. Decompressive laminectomy and durotomy did not improve motor function recovery, and rats in both of these treatment modalities developed neuropathic pain. Performing a durotomy also led to increased lesion volumes. Placement of DuraSeal was shown to cause compression in some rats in the duraplasty treatment groups. Decompressive duraplasty of 3 levels does not affect functional outcomes after injury but did increase white matter sparing. Decompressive duraplasty of 5 levels led to neuropathic pain development and increased lesion volumes. Further comparison of dural repair techniques is necessary.

Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides.

Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosorbent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELISA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of suramin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC50 between Bupleurum smithii var. parvifolium polysaccharides (1.055 mg/mL) and Bupleurum chinense polysaccharides (0.98 mg/mL) showed that, similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti-complementary properties against the lectin pathway. The results demonstrate that the described ELISA assay can compensate for the shortcomings of the hemolytic assay in lectin pathway.

N-Terminal signal sequence is required for cellular trafficking and hyaluronan-depolymerization of KIAA1199.

Recently, we disclosed that KIAA1199-mediated hyaluronan (HA) depolymerization requires an acidic cellular microenvironment (e.g. clathrin-coated vesicles or early endosomes), but no information about the structural basis underlying the cellular targeting and functional modification of KIAA1199 was available. Here, we show that the cleavage of N-terminal 30 amino acids occurs in functionally matured KIAA1199, and the deletion of the N-terminal portion results in altered intracellular trafficking of the molecule and loss of cellular HA depolymerization. These results suggest that the N-terminal portion of KIAA1199 functions as a cleavable signal sequence required for proper KIAA1199 translocation and KIAA1199-mediated HA depolymerization.

Induction of FKBP51 by aldosterone in intestinal epithelium.

Screening female rat distal colon preparations for aldosterone-induced genes identified the Hsp90-binding immunophilin FKBP51 as a major aldosterone-induced mRNA and protein. Limited induction of FKBP51 was observed also in other aldosterone-responsive tissues such as kidney medulla and heart. Ex vivo measurements in colonic tissue have characterized time course, dose response and receptor specificity of the induction of FKBP51. FKBP51 mRNA and protein were strongly up regulated by physiological concentrations of aldosterone in a late (greater than 2.5h) response to the hormone. Maximal increase in FKBP51 mRNA requires aldosterone concentrations that are higher than those needed to fully occupy the mineralocorticoid receptor (MR). Yet, the response is fully inhibited by the MR antagonist spironolactone and not inhibited and even stimulated by the glucocorticoid receptor (GR) antagonist RU486. These and related findings cannot be explained by a simple activation and dimerization of either MR or GR but are in agreement with response mediated by an MR-GR heterodimer. Overexpression or silencing FKBP51 in the kidney collecting duct cell line M1 had little or no effect on the aldosterone-induced increase in transepithelial Na(+) transport.

Spatial memory extinction: a c-Fos protein mapping study.

While the neuronal basis of spatial memory consolidation has been thoroughly studied, the substrates mediating the process of extinction remain largely unknown. This study aimed to evaluate the functional contribution of selected brain regions during the extinction of a previously acquired spatial memory task in the Morris water maze. For that purpose, we used adult male Wistar rats trained in a spatial reference memory task. Learning-related changes in c-Fos inmunoreactive cells after training were evaluated in cortical and subcortical regions. Results show that removal of the hidden platform in the water maze induced extinction of the previously reinforced escape behavior after 16 trials, without spontaneous recovery 24h later. Extinction was related with significantly higher numbers of c-Fos positive nuclei in amygdala nuclei and prefrontal cortex. On the other hand, the lateral mammillary bodies showed higher number of c-Fos positive cells than the control group. Therefore, in contrast with the results obtained in studies of classical conditioning, we show the involvement of diencephalic structures mediating this kind of learning. In summary, our findings suggest that medial prefrontal cortex, the amygdala complex and diencephalic structures like the lateral mammillary nuclei are relevant for the extinction of spatial memory.

Ventral midbrain neural stem cells have delayed neurogenic potential in vitro.

Neural stem cells (NSCs) have been the focus of an intensive effort to direct their differentiation in vitro towards desired neuronal phenotypes for cell replacement therapies. It is thought that NSCs derived from older embryos have limited neurogenic capacity and are restricted towards an astroglial fate. This idea is largely based on studies that typically analysed NSC-derived progeny following one week of in vitro differentiation. In this report, the neurogenic capacity of older ventral midbrain (VM) NSCs was assessed. When the older NSCs were differentiated for three weeks, there were significant increases in the numbers of newly born neurons at 14 and 21 days, as assessed by 5-bromo-2'-deoxyuridine (BrdU) incorporation. Therefore this study demonstrates that older NSCs retain significantly more neurogenic potential than was previously thought. These data have implications for NSC preparatory protocols and the choice of donor age for cell transplantation studies, and contributes to the understanding of NSC behaviour in vitro.

Control of antigen-binding to aluminum adjuvants and the immune response with a novel phosphonate linker.

The strongest mechanism for adsorption of antigens to aluminum adjuvants is ligand exchange, which involves the replacement of a surface hydroxyl on the adjuvant by a terminal phosphate group of the antigen. A novel phosphonate linker was developed that allows the addition of phosphonate (C-PO3) groups to proteins under controlled and chemically mild conditions. Increasing the number of linkers per protein molecule progressively increased the adsorption strength to aluminum hydroxide adjuvant (AH) as measured by elution in serum. The effect of phosphonate conjugation on the antibody response was determined with hen egg lysozyme (HEL), a protein that has the same charge as AH at neutral pH and does not adsorb to AH. The phosphonylated form of HEL (HEL-P) adsorbed to AH, indicating that the ligand exchange interaction could overcome the electrostatic repulsion. Mice injected with HEL-P/AH had a higher antibody titer to HEL than mice injected with HEL/AH, especially at lower antigen doses, suggesting that adsorption of antigen has a dose-sparing effect. Conjugation of CRM197, an antigen that adsorbs electrostatically to AH, with phosphonate linkers did not enhance the antibody response, indicating that adsorption by either electrostatic or ligand exchange to AH is sufficient to enhance the antibody response.

Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids.

GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at -60 mV in a dose-dependent manner with an EC50 of 32.3 µM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01 µM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn(2+) in a dose-dependent manner with an IC50 of 18 µM, whereas it was enhanced by 100 µM La(3+). The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, ß, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits.

Neuroprotective effects of NGF, BDNF, NT-3 and GDNF on axotomized extraocular motoneurons in neonatal rats.

Neurotrophic factors delivered from target muscles are essential for motoneuronal survival, mainly during development and early postnatal maturation. It has been shown that the disconnection between motoneurons and their innervated muscle by means of axotomy produces a vast neuronal death in neonatal animals. In the present work, we have evaluated the effects of different neurotrophic factors on motoneuronal survival after neonatal axotomy, using as a model the motoneurons innervating the extraocular eye muscles. With this purpose, neonatal rats were monocularly enucleated at the day of birth (postnatal day 0) and different neurotrophic treatments (NGF, BDNF, NT-3, GDNF and the mixture of BDNF+GDNF) were applied intraorbitally by means of a Gelfoam implant (a single dose of 5 µg of each factor). We first demonstrated that extraocular eye muscles of neonatal rats expressed these neurotrophic factors and therefore constituted a natural source of retrograde delivery for their innervating motoneurons. By histological and immunocytochemical methods we determined that all treatments significantly rescued extraocular motoneurons from axotomy-induced cell death. For the dose used, NGF and GDNF were the most potent survival factors for these motoneurons, followed by BDNF and lastly by NT-3. The simultaneous administration of BDNF and GDNF did not increase the survival-promoting effects above those obtained by GDNF alone. Interestingly, the rescue effects of all neurotrophic treatments persisted even 30 days after lesion. The administration of these neurotrophic factors, with the exception of NT-3, also prevented the loss of the cholinergic phenotype observed by 10 days after axotomy. At the dosage applied, NGF and GDNF were revealed again as the most effective neuroprotective agents against the axotomy-induced decrease in ChAT. Two remarkable findings highlighted in the present work that contrasted with other motoneuronal types after neonatal axotomy: first, the extremely high efficacy of NGF as a neuroprotective agent and, second, the long-lasting effects of neurotrophic administration on cell survival and ChAT expression in extraocular motoneurons.

Dynamic expression pattern of leucine-rich repeat neuronal protein 4 in the mouse dorsal root ganglia during development.

A member of leucine-rich repeat neuronal protein (Lrrn) family, Lrrn4, is a type I transmembrane protein and functions as a cell adhesion molecule. In our previous report, Lrrn4 is expressed in a subset of small-sized dorsal root ganglion (DRG) neurons of the adult mice. In the present study, we investigated the expression pattern of Lrrn4 in the developing DRGs. The expression of Lrrn4 was first observed in 7% of total DRG neurons at embryonic day (E) 13.5, gradually increasing to 44% at E17.5, reached the maximum level between E17.5 and postnatal day (P) 7, decreased drastically after P7, and became the adult level by P14. Interestingly, the expression of Lrrn4 was mainly observed in TrkC-positive neurons at E13.5, and the predominant expression was shifted from TrkC-positive neurons to TrkA-positive neurons between E15.5 and E17.5. As the central afferents of TrkC-positive and TrkA-positive neurons begin to penetrate into the spinal cord to form synapse with secondary neurons at E13.5 and E15.5, respectively, the time course of Lrrn4 expression may suggest the contribution of Lrrn4 to synaptic formation. In addition, some cell adhesion molecules containing leucine-rich repeat are identified as synaptic adhesion molecules, suggesting that the spatiotemporal expression pattern of Lrrn4 contributes to the development of synaptic function in the DRG neurons.

An inhibitory antibody blocks the first step in the dithiol/disulfide relay mechanism of the enzyme QSOX1.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a catalyst of disulfide bond formation that undergoes regulated secretion from fibroblasts and is over-produced in adenocarcinomas and other cancers. We have recently shown that QSOX1 is required for incorporation of particular laminin isoforms into the extracellular matrix (ECM) of cultured fibroblasts and, as a consequence, for tumor cell adhesion to and penetration of the ECM. The known role of laminins in integrin-mediated cell survival and motility suggests that controlling QSOX1 activity may provide a novel means of combating metastatic disease. With this motivation, we developed a monoclonal antibody that inhibits the activity of human QSOX1. Here, we present the biochemical and structural characterization of this antibody and demonstrate that it is a tight-binding inhibitor that blocks one of the redox-active sites in the enzyme, but not the site at which de novo disulfides are generated catalytically. Sulfhydryl oxidase activity is thus prevented without direct binding of the sulfhydryl oxidase domain, confirming the model for the interdomain QSOX1 electron transfer mechanism originally surmised based on mutagenesis and protein dissection. In addition, we developed a single-chain variant of the antibody and show that it is a potent QSOX1 inhibitor. The QSOX1 inhibitory antibody will be a valuable tool in studying the role of ECM composition and architecture in cell migration, and the recombinant version may be further developed for potential therapeutic applications based on manipulation of the tumor microenvironment.

Murine homologue of the human KIAA1199 is implicated in hyaluronan binding and depolymerization.

Recently, we have disclosed that human KIAA1199 (hKIAA1199) is a hyaluronan (HA) binding protein implicated in HA depolymerization. Although a murine homologue (mKiaa1199) was previously cloned, no information about the function of the molecule was available. Here, we show that cells transfected with mKiaa1199 cDNA selectively catabolized HA via the clathrin-coated pit pathway. A glycosaminoglycan-binding assay demonstrated the specific binding of mKiaa1199 to HA. These results were similar to our observations with hKIAA1199, although slight differences were found in the peak sizes of the minimum degradates of HA. We conclude that like hKIAA1199, mKiaa1199 is a hyaladherin, leading to HA depolymerization.

Spatial memory impairments in a prediabetic rat model.

Diabetes is associated with an increased risk for brain disorders, namely cognitive impairments associated with hippocampal dysfunction underlying diabetic encephalopathy. However, the impact of a prediabetic state on cognitive function is unknown. Therefore, we now investigated whether spatial learning and memory deficits and the underlying hippocampal dysfunction were already present in a prediabetic animal model. Adult Wistar rats drinking high-sucrose (HSu) diet (35% sucrose solution during 9 weeks) were compared to controls' drinking water. HSu rats exhibited fasting normoglycemia accompanied by hyperinsulinemia and hypertriglyceridemia in the fed state, and insulin resistance with impaired glucose tolerance confirming them as a prediabetic rodent model. HSu rats displayed a poorer performance in hippocampal-dependent short- and long-term spatial memory performance, assessed with the modified Y-maze and Morris water maze tasks, respectively; this was accompanied by a reduction of insulin receptor-ß density with normal levels of insulin receptor substrate-1 pSer636/639, and decreased hippocampal glucocorticoid receptor levels without changes of the plasma corticosterone levels. Importantly, HSu animals exhibited increased hippocampal levels of AMPA and NMDA receptor subunits GluA1 and GLUN1, respectively, whereas the levels of protein markers related to nerve terminals (synaptophysin) and oxidative stress/inflammation (HNE, RAGE, TNF-α) remained unaltered. These findings indicate that 9 weeks of sucrose consumption resulted in a metabolic condition suggestive of a prediabetic state, which translated into short- and long-term spatial memory deficits accompanied by alterations in hippocampal glutamatergic neurotransmission and abnormal glucocorticoid signaling.

Apotransferrin-induced recovery after hypoxic/ischaemic injury on myelination.

We have previously demonstrated that aTf (apotransferrin) accelerates maturation of OLs (oligodendrocytes) in vitro as well as in vivo. The purpose of this study is to determine whether aTf plays a functional role in a model of H/I (hypoxia/ischaemia) in the neonatal brain. Twenty-four hours after H/I insult, neonatal rats were intracranially injected with aTf and the effects of this treatment were evaluated in the CC (corpus callosum) as well as the SVZ (subventricular zone) at different time points. Similar to previous studies, the H/I event produced severe demyelination in the CC. Demyelination was accompanied by microglial activation, astrogliosis and iron deposition. Ferritin levels increased together with lipid peroxidation and apoptotic cell death. Histological examination after the H/I event in brain tissue of aTf-treated animals (H/I aTF) revealed a great number of mature OLs repopulating the CC compared with saline-treated animals (H/I S). ApoTf treatment induced a gradual increase in MBP (myelin basic protein) and myelin lipid staining in the CC reaching normal levels after 15 days. Furthermore, significant increase in the number of OPCs (oligodendroglial progenitor cells) was found in the SVZ of aTf-treated brains compared with H/I S. Specifically, there was a rise in cells positive for OPC markers, i.e. PDGFRα and SHH(+) cells, with a decrease in cleaved-caspase-3(+) cells compared with H/I S. Additionally, neurospheres from aTf-treated rats were bigger in size and produced more O4/MBP(+) cells. Our findings indicate a role for aTf as a potential inducer of OLs in neonatal rat brain in acute demyelination caused by H/I and a contribution to the differentiation/maturation of OLs and survival/migration of SVZ progenitors after demyelination in vivo.