Multiple sclerosis is a chronic demyelinating disease of the central nervous system. Spontaneous remyelination during early disease stages is thought to preserve and partially restore function. However, this process ceases in later stages despite the presence of pre-oligodendrocytes. Cuprizone-induced demyelination is a useful model with which to study the remyelination process. Previous studies have demonstrated heterogeneities in demyelination in individual animals. Here we investigated regional differences in demyelination and remyelination within the corpus callosum. C57BL/6 mice were fed 0.2% cuprizone for 5 weeks to induce demyelination. Remyelination was examined 2-5 weeks after cuprizone withdrawal. Immunohistochemistry and electron microscopy were used to quantify regional differences in demyelination, gliosis, and remyelination. We found that, while demyelination was limited in the rostral region of corpus callosum, nearly complete demyelination occurred in the caudal callosum, beginning at approximately -0.5mm from bregma. Astrogliosis and microgliosis were correlated with demyelination and differed between the rostral and caudal callosal structures. Remyelination upon cessation of cuprizone ensued at different rates with splenium remyelinating faster than dorsal hippocampal commissure. Our data show anatomical differences of cuprizone-induced demyelination and remyelination in the corpus callosum and the importance of examining specific callosal regions in myelin repair studies using this model.

14-3-3 proteins are found in all eukaryotes where they act as regulators of diverse signalling pathways associated with a wide range of biological processes. In this study the functional characterization of the ZmGF14-6 gene encoding a maize 14-3-3 protein is reported. Gene expression analyses indicated that ZmGF14-6 is up-regulated by fungal infection and salt treatment in maize plants, whereas its expression is down-regulated by drought stress. It is reported that rice plants constitutively expressing ZmGF14-6 displayed enhanced tolerance to drought stress which was accompanied by a stronger induction of drought-associated rice genes. However, rice plants expressing ZmGF14-6 either in a constitutive or under a pathogen-inducible regime showed a higher susceptibility to infection by the fungal pathogens Fusarium verticillioides and Magnaporthe oryzae. Under infection conditions, a lower intensity in the expression of defence-related genes occurred in ZmGF14-6 rice plants. These findings support that ZmGF14-6 positively regulates drought tolerance in transgenic rice while negatively modulating the plant defence response to pathogen infection. Transient expression assays of fluorescently labelled ZmGF14-6 protein in onion epidermal cells revealed a widespread distribution of ZmGF14-6 in the cytoplasm and nucleus. Additionally, colocalization experiments of fluorescently labelled ZmGF14-6 with organelle markers, in combination with cell labelling with the endocytic tracer FM4-64, revealed a subcellular localization of ZmGF14-6 in the early endosomes. Taken together, these results improve our understanding of the role of ZmGF14-6 in stress signalling pathways, while indicating that ZmGF14-6 inversely regulates the plant response to biotic and abiotic stresses.

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30 months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.

The transcription factor, Krüppel-like factor 4 (KLF4), plays an important role in regulating the proliferation of vascular smooth muscle cells. This study aimed to examine the effect of rapamycin on the expression of KLF4 and the role of KLF4 in arterial neointimal formation.

Given strong regional specialization of the brain, cerebral angiogenesis may be regionally modified during normal aging. To test this hypothesis, expression of a broad cadre of angiogenesis-associated genes was assayed at the neurovascular unit (NVU) in discrete brain regions of young versus aged mice by laser capture microdissection coupled to quantitative real-time polymerase chain reaction (PCR). Complementary quantitative capillary density/branching studies were performed as well. Effects of physical exercise were also assayed to determine if age-related trends could be reversed. Additionally, gene response to hypoxia was probed to highlight age-associated weaknesses in adapting to this angiogenic stress. Aging impacted resting expression of angiogenesis-associated genes at the NVU in a region-dependent manner. Physical exercise reversed some of these age-associated gene trends, as well as positively influenced cerebral capillary density/branching in a region-dependent way. Lastly, hypoxia revealed a weaker angiogenic response in aged brain. These results suggest heterogeneous changes in angiogenic capacity of the brain during normal aging, and imply a therapeutic benefit of physical exercise that acts at the level of the NVU.

The interpeduncular nucleus (IP) is a key limbic structure, highly conserved evolutionarily among vertebrates. The IP receives indirect input from limbic areas of the telencephalon, relayed by the habenula via the fasciculus retroflexus. The function of the habenulo-IP complex is poorly understood, although there is evidence that in rodents it modulates behaviors such as learning and memory, avoidance, reward and affective states. The IP has been an important subject of interest for neuroscientists, and there are multiple studies about the adult structure, chemoarchitecture and its connectivity, with complex results, due to the presence of multiple cell types across a variety of subnuclei. However, the ontogenetic origins of these populations have not been examined, and there is some controversy about its location in the midbrain-anterior hindbrain area. To address these issues, we first investigated the anteroposterior (AP) origin of the IP complex by fate-mapping its neuromeric origin in the chick, discovering that the IP develops strictly within isthmus and rhombomere 1. Next, we studied the dorsoventral (DV) positional identity of subpopulations of the IP complex. Our results indicate that there are at least four IP progenitor domains along the DV axis. These specific domains give rise to distinct subtypes of cell populations that target the IP with variable subnuclear specificity. Interestingly, these populations can be characterized by differential expression of the transcription factors Pax7, Nkx6.1, Otp, and Otx2. Each of these subpopulations follows a specific route of migration from its source, and all reach the IP roughly at the same stage. Remarkably, IP progenitor domains were found both in the alar and basal plates. Some IP populations showed rostrocaudal restriction in their origins (isthmus versus anterior or posterior r1 regions). A tentative developmental model of the structure of the avian IP is proposed. The IP emerges as a plurisegmental and developmentally heterogeneous formation that forms ventromedially within the isthmus and r1. These findings are relevant since they help to understand the highly complex chemoarchitecture, hodology and functions of this important brainstem structure.

The developmental relationship between the posterior embryonic and extraembryonic regions of the mammalian gastrula is poorly understood. Although many different cell types are deployed within this region, only the primordial germ cells (PGCs) have been closely studied. Recent evidence has suggested that the allantois, within which the PGCs temporarily take up residence, contains a pool of cells, called the Allantoic Core Domain (ACD), critical for allantoic elongation to the chorion. Here, we have asked whether the STELLA-positive cells found within this region, thought to be specified PGCs, are actually part of the ACD and to what extent they, and other ACD cells, contribute to the allantois and fetal tissues. To address these hypotheses, STELLA was immunolocalized to the mouse gastrula between Early Streak (ES) and 12-somite pair (-s) stages (~6.75-9.0 days post coitum, dpc) in histological sections. STELLA was found in both the nucleus and cytoplasm in a variety of cell types, both within and outside of the putative PGC trajectory. Fate-mapping the headfold-stage (~7.75-8.0 dpc) posterior region, by which time PGCs are thought to be segregated into a distinct lineage, revealed that the STELLA-positive proximal ACD and intraembryonic posterior primitive streak (IPS) contributed to a wide range of somatic tissues that encompassed derivatives of the three primary germ layers. This contribution included STELLA-positive cells localizing to tissues both within and outside of the putative PGC trajectory. Thus, while STELLA may identify a subpopulation of cells destined for the PGC lineage, our findings reveal that it may be part of a broader niche that encompasses the ACD and through which the STELLA population may contribute cells to a wide variety of posterior tissues of the mouse gastrula.

A virgin ant queen has only one opportunity in her lifetime to realize her reproductive fitness when she leaves her nest for a mating flight. After successful mating she sheds her wings, excavates a nest and starts laying eggs to initiate her own colony. Here we report the results of our study on two related species of Camponotus ants - day active Camponotus paria and night active Camponotus compressus - aimed at investigating (i) if there exist inter-species differences in the activity and phototactic behaviors of males and queens, (ii) whether these behaviors in the queen change after mating, and (iii) whether the activity rhythm of queens changes with age. We find that while activity profiles differ between C. paria and C. compressus virgin males and queens, such differences in queens disappear after mating. Once mated, the activity rhythm of queens shows little change with age; the rhythm in virgin queens, on the other hand, changes considerably. As virgins, C. paria queens are positively phototactic, while C. compressus queens are negatively phototactic. After mating, C. paria queens become less phototactic, particularly during the subjective night, while C. compressus queens remain negatively phototactic. These results indicate that there are considerable differences in the activity and phototactic behaviors of virgin queens of the two related species of Camponotus ants. Most of these differences disappear after mating, which suggests that these behaviors may have evolved primarily for the proper execution of pre-mating events.

Extensive alternative splicing is observed in the mammalian nervous system providing for protein diversity and specificity to accomplish the complex neuronal functions. Mechanisms underlying neuron specific splicing are not yet well understood. Among the factors regulating splicing of major importance are serine/arginine protein kinases (SRPKs) that phosphorylate SR splicing factors. SRPK1 is known to be expressed in the mammalian central nervous system. The present immunohistochemical study reveals a region- and neuron-specific localization of SRPK1 in human brain. The potential involvement of the kinase in the regulation of alternative splicing of various neuronal proteins is discussed.

Oxidatively damaged DNA base lesions are considered to be mainly repaired by 8-oxoguanine DNA glycosylase (OGG1) mediated pathways. We investigated the effect of the OGG1 Ser326Cys polymorphism on the level and repair of oxidatively damaged DNA in mononuclear blood cells (MNBC) by means of the comet assay. We collected blood samples from 1,019 healthy subjects and genotyped for the OGG1 Ser326Cys polymorphism. We found 49 subjects homozygous for the variant genotype (Cys/Cys) and selected same numbers of age-matched subjects with the heterozygous (Ser/Cys) and homozygous wild-type genotype (Ser/Ser). Carriers of the Cys/Cys genotype had higher levels of formamidopyrimidine DNA glycosylase (FPG) sensitive sites in MNBC (0.31 ± 0.03 lesions/10(6)bp) compared to Ser/Ser (0.19 ± 0.02 lesions/10(6)bp, P<0.01). The level of hOGG1 sensitive sites in MNBC from the Ser326Cys carriers (0.19 ± 0.16 lesions/10(6) bp) was also higher compared to the Ser/Ser genotype (0.11 ± 0.09 lesions/10(6) bp, P<0.05). Still, there was no genotype-related difference in DNA repair incision activity of MNBC extracts on nucleoids with oxidatively damaged DNA induced by Ro19-8022/white light (P=0.20). In addition, there were no differences in the expression of OGG1 (P=0.69), ERCC1 (P=0.62), MUTYH (P=0.85), NEIL1 (P=0.17) or NUDT1 (P=0.48) in whole blood. Our results indicate that the OGG1 Ser326Cys polymorphism has limited influence on the DNA repair incisions by extracts of MNBC, whereas the apparent increased risk of cancer in subjects with the Cys/Cys genotype may be because of higher levels of oxidatively damaged DNA.

The epidermal growth factor (EGF) activates the phosphatidylinositol 3-kinase (PI3K)-Akt cascade among other signaling pathways. This route is involved in cell proliferation and survival, therefore, its dysregulation can promote cancer. Considering the relevance of the PI3K-Akt signaling in cell survival and in the pathogenesis of cancer, and that GH was reported to modulate EGFR expression and signaling, the objective of this study was to analyze the effects of increased GH levels on EGF-induced PI3K-Akt signaling. EGF-induced signaling was evaluated in the liver of GH-overexpressing transgenic mice and in their normal siblings. While Akt expression was increased in GH-overexpressing mice, EGF-induced phosphorylation of Akt, relative to its protein content, was diminished at Ser473 and inhibited at Thr308; consequently, mTOR, which is a substrate of Akt, was not activated by EGF. However, the activation of PDK1, a kinase involved in Akt phosphorylation at Thr308, was not reduced in transgenic mice. Kinetics studies of EGF-induced Akt phosphorylation showed that it is rapidly and transiently induced in GH-overexpressing mice compared with normal siblings. Thus, the expression and activity of phosphatases involved in the termination of the PI3K-Akt signaling were studied. In transgenic mice, neither PTEN nor PP2A were hyperactivated; however, EGF induced the rapid and transient association of SHP-2 to Gab1, which mediates association to EGFR and activation of PI3K. Rapid recruitment of SHP2, which would accelerate the termination of the proliferative signal induced, could be therefore contributing to the diminished EGF-induced activity of Akt in GH-overexpressing mice.

Transferrin receptor-1 (TfR1) is a cell membrane-associated glycoprotein responsible for incorporation of the iron bound to transferrin through an endocytotic process from the circulating blood. Iron is believed to play a dual role as an active center of the electron transfer system in mitochondria and as an endogenous cytotoxin through promoted generation of reactive oxygen species in different eukaryotic cells. In this study, we evaluated expression profiles of different genes related to iron mobilization across plasma membranes in neuronal cells. Marked mRNA expression was seen for various iron-related genes such as TfR1 in cultured mouse neocortical neurons, while TfR1 mRNA levels were more than doubled during culture from 3 to 6days. In mouse embryonal carcinoma P19 cells endowed to differentiate into neuronal and astroglial lineages, a transient increase was seen in both mRNA and corresponding protein for TfR1 in association with neuronal marker expression during culture with all-trans retinoic acid (ATRA). In neuronal Neuro2A cells cultured with ATRA, moreover, neurite was elongated together with increased expression of both mRNA and protein for TfR1. Overexpression of TfR1 significantly decreased the length of neurite elongated, however, while significant promotion was invariably seen in the neurite elongation in Neuro2A cells transfected with TfR1 siRNA as well as in Neuro2A cells cultured with an iron chelator. These results suggest that TfR1 would be highly expressed by neurons rather than astroglia to play a negative role in the neurite outgrowth after the incorporation of circulating transferrin in the brain.

Schizophrenia is a severe neuropsychiatric disorder with high heritability, though its exact etiopathogenesis is yet unknown. An increasing number of studies point to the importance of white matter anomalies in the pathophysiology of schizophrenia. While several studies have identified the impact of schizophrenia susceptibility gene variants on gray matter anatomy in both schizophrenia patients and healthy risk variant carriers, studies dealing with the impact of these gene variants on white matter integrity are still scarce. We here present a study on the effects of a Dysbindin schizophrenia susceptibility gene variant on fiber tract integrity in healthy young subjects. 101 subjects genotyped for Dysbindin-gene variant rs1018381, though without personal or first degree relative history of psychiatric disorders underwent diffusion tensor imaging (DTI), 83 of them were included in the final analysis. We used Tract-Based Spatial Statistics (TBSS) analysis to delineate the major fiber tracts. Carriers of the minor allele T of the rs1018381 in the Dysbindin gene showed two clusters of reduced fractional anisotropy (FA) values in the perihippocampal region of the right temporal lobe compared to homozygote carriers of the major allele C. Clusters of increased FA values in T-allele carriers were found in the left prefrontal white matter, the right fornix, the right midbrain area, the left callosal body, the left cerebellum and in proximity of the right superior medial gyrus. Dysbindin has been implicated in neurite outgrowth and morphology. Impairments in anatomic connectivity as found associated with the minor Dysbindin allele in our study may result in increased risk for schizophrenia due to altered fiber tracts.

Diffusion tensor imaging (DTI) is widely used to characterize tissue micro-architecture and brain connectivity. In regions of crossing fibers, however, the tensor model fails because it cannot represent multiple, independent intra-voxel orientations. Most of the methods that have been proposed to resolve this problem require diffusion magnetic resonance imaging (MRI) data that comprise large numbers of angles and high b-values, making them problematic for routine clinical imaging and many scientific studies. We present a technique based on compressed sensing that can resolve crossing fibers using diffusion MRI data that can be rapidly and routinely acquired in the clinic (30 directions, b-value equal to 700 s/mm2). The method assumes that the observed data can be well fit using a sparse linear combination of tensors taken from a fixed collection of possible tensors each having a different orientation. A fast algorithm for computing the best orientations based on a hierarchical compressed sensing algorithm and a novel metric for comparing estimated orientations are also proposed. The performance of this approach is demonstrated using both simulations and in vivo images. The method is observed to resolve crossing fibers using conventional data as well as a standard q-ball approach using much richer data that requires considerably more image acquisition time.

The intrahepatic biliary ducts transport bile produced by the hepatocytes out of the liver. Defects in biliary cell differentiation and biliary duct remodeling cause a variety of congenital diseases including Alagille Syndrome and polycystic liver disease. While the molecular pathways regulating biliary cell differentiation have received increasing attention (Lemaigre, 2010), less is known about the cellular behavior underlying biliary duct remodeling. Here, we have identified a novel gene, claudin 15-like b (cldn15lb), which exhibits a unique and dynamic expression pattern in the hepatocytes and biliary epithelial cells in zebrafish. Claudins are tight junction proteins that have been implicated in maintaining epithelial polarity, regulating paracellular transport, and providing barrier function. In zebrafish cldn15lb mutant livers, tight junctions are observed between hepatocytes, but these cells show polarization defects as well as canalicular malformations. Furthermore, cldn15lb mutants show abnormalities in biliary duct morphogenesis whereby biliary epithelial cells remain clustered together and form a disorganized network. Our data suggest that Cldn15lb plays an important role in the remodeling process during biliary duct morphogenesis. Thus, cldn15lb mutants provide a novel in vivo model to study the role of tight junction proteins in the remodeling of the biliary network and hereditary cholestasis.

The tandem starch-binding domains (KvSBD) located at carboxy-terminal region of halophilic α-amylase from moderate halophile, Kocuria varians, were expressed in E. coli with amino-terminal hexa-His-tag and purified to homogeneity. The recombinant KvSBD showed binding activity to raw starch granules at low to high salt concentrations. The binding activity of KvSBD to starch was fully reversible after heat-treatment at 85°C. Circular dichroism and thermal scanning experiments indicated that KvSBD showed fully reversible refolding upon cooling after complete melting at 70°C in the presence of 0.2-2.0M NaCl. The refolding rate was enhanced with higher salt concentration.

Progesterone and its metabolite, allopregnanolone, are neurosteroids that are present at high concentrations in fetal brains that decrease right after birth. Allopregnanolone is a potent positive modulator of γ-aminobutyric acid A (GABA(A)) receptor function. We examined the effect of exogenous administration of these steroids on hypoxic-ischemic encephalopathy in immature rats. Progesterone (10mg/kg), allopregnanolone (10mg/kg), or vehicle alone was intraperitoneally administered immediately before and then subcutaneously 6h and 24h after hypoxia-ischemia to postnatal day 7 (P7), day 14 (P14), and day 21 (P21) rats. The effects of the treatments were evaluated using histological analyses (hemispheric volumes and semi-quantitative scoring for neuropathologic injury). Both progesterone and allopregnanolone significantly exacerbated brain injury in P7 and P14 rats, but not in P21 rats. This detrimental effect was similar across the examined brain regions (the cortex, striatum, hippocampus, and thalamus) and showed no sex differences. Co-administration of the GABA(A) receptor antagonist, bicuculline, partially mitigated the exacerbating effect of allopregnanolone. Based on the similarity of the effects of these neurosteroids, we speculate that progesterone accentuates neuronal injury mainly via the activity of allopregnanolone. The present study indicates that the detrimental effects of allopregnanolone were, at least in part, mediated via GABAergic neuroexcitability. This is in line with the notion that GABA is excitatory for immature neurons, while it is inhibitory for mature neurons.

Here we show that pemetrexed-treated mesothelioma cells undergo accelerated senescence. This is characterized by the secretion of proinflammatory and mitogenic cytokines, reminiscent of an SASP (senescence-associated secretory phenotype). Conditioned media from senescent MPM (malignant pleural mesothelioma) cells trigger the emergence of EMT (epithelial-to-mesenchymal)-like, clonogenic and chemoresistant cell subpopulations, expressing high levels of ALDH (aldehyde dehydrogenase) activity (ALDH(bright) cells). We show by fluorescence-activated cell sorting of purified ALDH(bright) and ALDH(low) cells, that both cell-autonomous and cell-non-autonomous mechanisms converge to maintain the SASP-induced, EMT-like cell subpopulations. Chemoresistant ALDH(bright) cells exist within primary MPM specimens and enrichment for ALDH(bright) cells correlates with an earlier tumor onset into NOD/SCID mice. We show that RAS(v12) expression induces SASP-like changes in untransformed human mesothelial cells, and that p53 ablation increases the effect of RAS(v12) expression. We identify STAT3 activation as a crucial event downstream to SASP signaling. In fact, small hairpin RNA-mediated ablation of STAT3 deeply attenuates the induction of EMT genes and the increase of ALDH(bright) cells induced by SASP-cytokines. This strongly affects the chemoresistance of MPM cells in vitro and leads to anticancer effects in vivo.

Expression of CD200, the gene encoding the ligand for the inhibitory immune receptor CD200R, is an independent prognostic factor for various forms of leukemia predicting worse overall survival of the patients. The enhanced expression of CD200 on the tumors implies that anti-tumor responses can be enhanced by blockage of the CD200-CD200R interaction. Indeed, antibody-mediated blockade of the CD200-CD200R inhibitory axis is currently evaluated in clinical tests to boost immune responses against CD200-expressing tumors. Here, we show that mice lacking CD200, the exclusive ligand for CD200R, are resistant to chemical skin carcinogenesis. Importantly, CD200R controls tumor outgrowth independently of CD200 expression by the tumor cells themselves. Furthermore, Cd200(-/-) mice do not become tolerant to intranasally administered antigens, suggesting that tumor rejection is normally suppressed through CD200-induced immune tolerance. Decreased tumor outgrowth is accompanied by increased expression of the proinflammatory cytokines interleukin (IL)-1β and IL-6 by the lymph node (LN) dendritic cells. During carcinogenesis, skin-draining LNs of Cd200(-/-) mice contain increased numbers of IL-17-producing FoxP3(+) cells, which preferentially home to the tumors. Thus, the CD200-CD200R axis induces tolerance to external and tumor antigens and influences the T-regulatory/Th17 cell ratio. We demonstrate for the first time that the absence of CD200R signaling inhibits outgrowth of an endogenous tumor irrespective of CD200 expression by the tumor cells. This important paradigm shift leads to a much broader applicability of CD200-blockade in the treatment of tumors.

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer's disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1(M146L)/APP(751SL) mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin-cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.