Multiple sclerosis: Fas signaling in oligodendrocyte cell death.

Fas is a cell surface receptor that transduces cell death signals when cross-linked by agonist antibodies or by fas ligand. In this study, we examined the potential of fas to contribute to oligodendrocyte (OL) injury and demyelination as they occur in the human demyelinating disease multiple sclerosis (MS). Immunohistochemical study of central nervous system (CNS) tissue from MS subjects demonstrated elevated fas expression on OLs in chronic active and chronic silent MS lesions compared with OLs in control tissue from subjects with or without other neurologic diseases. In such lesions, microglia and infiltrating lymphocytes displayed intense immunoreactivity to fas ligand. In dissociated glial cell cultures prepared from human adult CNS tissue, fas expression was restricted to OLs. Fas ligation with the anti-fas monoclonal antibody M3 or with the fas-ligand induced rapid OL cell membrane lysis, assessed by LDH release and trypan blue uptake and subsequent cell death. In contrast to the activity of fas in other cellular systems, dying OLs did not exhibit evidence of apoptosis, assessed morphologically and by terminal transferase-mediated d-uridine triphosphate-biotin nick-end-labeling staining for DNA fragmentation. Other stimuli such as C2-ceramide were capable of inducing rapid apoptosis in OLs. Antibodies directed at other surface molecules expressed on OLs or the M33 non-activating anti-fas monoclonal antibody did not induce cytolysis of OLs. Our results suggest that fas-mediated signaling might contribute in a novel cytolytic manner to immune-mediated OL injury in MS.

A 14,000-year oxygen isotope record from diatom silica in two alpine lakes on Mt. Kenya.

Oxygen isotopes are sensitive tracers of climate change in tropical regions. Abrupt shifts of up to 18 per mil in the oxygen isotope ratio of diatom silica have been found in a 14,000-year record from two alpine lakes on Mt. Kenya. Interpretation of tropical-montane isotope records is controversial, especially concerning the relative roles of precipitation and temperature. Here, we argue that Holocene variations in delta(18)O are better explained by lake moisture balance than by temperature-induced fractionation. Episodes of heavy convective precipitation dated approximately 11,100 to 8600, 6700 to 5600, 2900 to 1900, and <1300 years before the present were linked to enhanced soil erosion, neoglacial ice advances, and forest expansion on Mt. Kenya.

Strong and recurring seasonality revealed within stream diatom assemblages.

Improving stream water quality in agricultural landscapes is an ecological priority and a legislative duty for many governments. Ecosystem health can be effectively characterised by organisms sensitive to water quality changes such as diatoms, single-celled algae that are a ubiquitous component of stream benthos. Diatoms respond within daily timescales to variables including light, temperature, nutrient availability and flow conditions that result from weather and land use characteristics. However, little consideration has been given to the ecological dynamics of diatoms through repeated seasonal cycles when assessing trajectories of stream function, even in catchments actively managed to reduce human pressures. Here, six years of monthly diatom samples from three independent streams, each receiving differing levels of diffuse agricultural pollution, reveal robust and repeated seasonal variation. Predicted seasonal changes in climate-related variables and anticipated ecological impacts must be fully captured in future ecological and water quality assessments, if the apparent resistance of stream ecosystems to pollution mitigation measures is to be better understood.

Disruption of NGF binding to the low affinity neurotrophin receptor p75LNTR reduces NGF binding to TrkA on PC12 cells.

The role of the low affinity neurotrophin receptor, p75LNTR, in NGF-mediated signal transduction has been examined. Our results show that treatment of PC12 cells with MC192, a monoclonal antibody directed against p75LNTR, results in reduced NGF binding to TrkA and attenuated TrkA activation. Use of mutant NGF that binds TrkA but not p75LNTR shows that the MC192 effect requires that NGF bind the p75LNTR receptor. To explore the possibility that MC192 disrupts some normal functional role of p75LNTR, BDNF was used to block binding of NGF to p75LNTR on PC12 cells. By preventing NGF binding to p75LNTR, NGF binding to TrkA and NGF-mediated signal transduction were reduced. We propose that p75LNTR normally acts to increase binding of NGF to TrkA, possibly by increasing the local NGF concentration in the microenvironment surrounding the cell surface TrkA receptor.

Detection of brain-derived neurotrophic factor-like activity in fibroblasts and Schwann cells: inhibition by antibodies to NGF.

mRNA coding for brain-derived neurotrophic factor (BDNF) has been detected in cultured L929 fibroblasts, rat dermal fibroblasts, and sciatic nerve Schwann cells, as well as in rat skin. Medium conditioned by cultured fibroblasts and Schwann cells also stimulates neurite growth from retinal explants and promotes the survival in culture of BDNF-responsive sensory neurons; biological activity is abolished by antibodies raised against NGF. These results suggest that molecules with BDNF-like activity may be produced by cells in the peripheral nervous system and that the BDNF-like activity in fibroblasts and Schwann cells is derived from molecules immunologically related to NGF. In support of this concept, antibodies against NGF have been found to reduce the biological activity of recombinant BDNF in culture and to cross-react with BDNF on Western blots.

NRAGE, a novel MAGE protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis.

The mechanisms employed by the p75 neurotrophin receptor (p75NTR) to mediate neurotrophin-dependent apoptosis are poorly defined. Two-hybrid analyses were used to identify proteins involved in p75NTR apoptotic signaling, and a p75NTR binding partner termed NRAGE (for neurotrophin receptor-interacting MAGE homolog) was identified. NRAGE binds p75NTR in vitro and in vivo, and NRAGE associates with the plasma membrane when NGF is bound to p75NTR. NRAGE blocks the physical association of p75NTR with TrkA, and, conversely, TrkA overexpression eliminates NRAGE-mediated NGF-dependent death, indicating that interactions of NRAGE or TrkA with p75NTR are functionally and physically exclusive. NRAGE overexpression facilitates cell cycle arrest and permits NGF-dependent apoptosis within sympathetic neuron precursors cells. Our results show that NRAGE contributes to p75NTR-dependent cell death and suggest novel functions for MAGE family proteins.

Major agricultural changes required to mitigate phosphorus losses under climate change.

Phosphorus losses from land to water will be impacted by climate change and land management for food production, with detrimental impacts on aquatic ecosystems. Here we use a unique combination of methods to evaluate the impact of projected climate change on future phosphorus transfers, and to assess what scale of agricultural change would be needed to mitigate these transfers. We combine novel high-frequency phosphorus flux data from three representative catchments across the UK, a new high-spatial resolution climate model, uncertainty estimates from an ensemble of future climate simulations, two phosphorus transfer models of contrasting complexity and a simplified representation of the potential intensification of agriculture based on expert elicitation from land managers. We show that the effect of climate change on average winter phosphorus loads (predicted increase up to 30% by 2050s) will be limited only by large-scale agricultural changes (e.g., 20-80% reduction in phosphorus inputs).The impact of climate change on phosphorus (P) loss from land to water is unclear. Here, the authors use P flux data, climate simulations and P transfer models to show that only large scale agricultural change will limit the effect of climate change on average winter P loads in three catchments across the UK.

CD95-CD95L: can the brain learn from the immune system?

Members of the tumor necrosis factor/nerve growth factor receptor superfamily of cell-surface molecules can play the dual role of mediating either cytotoxicity or cell survival, both in the immune system and in the nervous system. A member of this superfamily, CD95 (also known as ApoI or Fas), was initially identified in the immune system and has been shown to mediate receptor-dependent programmed cell death and to be expressed in the nervous system. In neurodegenerative disorders, CD95-CD95 ligand expression on glial cells might precede receptor-mediated apoptosis by cells of the CNS. It is now being recognized that CD95 signaling by immune cells mediates effects other than apoptosis, such as cell survival and under inflammatory conditions expression of this protein promotes neural-immune interactions. Both neuroscientists and immunologists can contribute to defining the mechanisms underlying these divergent effects and utilize such knowledge to aid understanding of cell death and survival.

Eiger-induced cell death relies on Rac1-dependent endocytosis.

Signaling via tumor necrosis factor receptor (TNFR) superfamily members regulates cellular life and death decisions. A subset of mammalian TNFR proteins, most notably the p75 neurotrophin receptor (p75NTR), induces cell death through a pathway that requires activation of c-Jun N-terminal kinases (JNKs). However the receptor-proximal signaling events that mediate this remain unclear. Drosophila express a single tumor necrosis factor (TNF) ligand termed Eiger (Egr) that activates JNK-dependent cell death. We have exploited this model to identify phylogenetically conserved signaling events that allow Egr to induce JNK activation and cell death in vivo. Here we report that Rac1, a small GTPase, is specifically required in Egr-mediated cell death. rac1 loss of function blocks Egr-induced cell death, whereas Rac1 overexpression enhances Egr-induced killing. We identify Vav as a GEF for Rac1 in this pathway and demonstrate that dLRRK functions as a negative regulator of Rac1 that normally acts to constrain Egr-induced death. Thus dLRRK loss of function increases Egr-induced cell death in the fly. We further show that Rac1-dependent entry of Egr into early endosomes is a crucial prerequisite for JNK activation and for cell death and show that this entry requires the activity of Rab21 and Rab7. These findings reveal novel regulatory mechanisms that allow Rac1 to contribute to Egr-induced JNK activation and cell death.

p75 neurotrophin receptor expression is induced in apoptotic neurons after seizure.

Seizure causes neuronal cell loss in both animal models and human epilepsy. To determine the contribution of apoptotic mechanisms to seizure-induced neuronal cell death, rat brains were examined for the occurrence of terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL)-positive nuclei after pilocarpine-induced seizure. Numerous TUNEL-positive cells were observed throughout the postseizure hippocampus, piriform cortex, and entorhinal cortex. Combined TUNEL/NeuN immunocytochemistry demonstrated that the vast majority of TUNEL-positive cells were neurons. To identify components of the signal transduction cascade promoting postseizure apoptosis, the expression of the p75 neurotrophin receptor (p75NTR) was examined. Seizure-induced increases in p75NTR protein and mRNA were detected in hippocampus, piriform cortex, and entorhinal cortex. Immunohistochemical double labeling revealed almost complete correspondence between TUNEL-positive and p75NTR-expressing cells, suggesting that seizure-induced neuronal loss within the CNS occurs through apoptotic signaling cascades involving p75NTR.

Subcellular localization of full-length and truncated Trk receptor isoforms in polarized neurons and epithelial cells.

Neurotrophins affect neuronal development and plasticity via spatially localized effects, yet little is known about the subcellular distribution of the Trk neurotrophin receptors and the impact of this distribution on neurotrophin action. To address this, we examined the subcellular location of full-length TrkB and TrkC tyrosine kinase receptors and truncated TrkB isoforms after transfection of Madin-Darby canine kidney (MDCK) cells, dissociated primary hippocampal neurons, and cortical neurons within intact brain slices. Myc-, herpes virus glycoprotein (HVG)-, or FLAG-derived epitope-tagged receptor isoforms were created to allow their unambiguous identification and localization after transfection. All tagged receptors were appropriately synthesized, and full-length myc-TrkB and myc-TrkC mediated appropriate neurotrophin-signaling events. We found that full-length TrkB receptors were excluded from the apical domain of MDCK cells but that TrkC receptors were present in both apical and basolateral domains. Full-length TrkB and TrkC were found throughout transfected primary cultured hippocampal neurons and transfected neurons in neocortical brain slices and showed no evidence of vectorial sorting. Truncated forms of TrkB were also homogeneously distributed in MDCK cells, dissociated hippocampal neurons, and cortical neurons within slice preparations. Levels of full-length and truncated TrkB were examined in postsynaptic densities; both receptor isoforms were present but only moderately enriched in these structures. Together, these findings suggest that Trk receptors are uniformly distributed in both axonal and dendritic compartments and that local neurotrophin responses are controlled by other mechanisms.

p75NTR: A study in contrasts.

The p75 neurotrophin receptor (p75NTR) and trkA, trkB and trkC mediate the physiological effects of the neurotrophins. The trk receptors are responsible for the stereotypical survival and growth properties of the neurotrophins but defining the physiological function of the p75NTR has proven difficult. The p75NTR binds each of the neurotrophins with low nanomolar affinity whereas the three trk receptors show strong binding preferences for individual neurotrophins; in some cell types, p75NTR is the only neurotrophin receptor whereas in others it is co-expressed with the trks. The analysis of p75NTR function has been complicated by the fact that the predominant physiological role of p75NTR changes dramatically depending on cell context. Available data suggests that in cells where p75NTR is co-expressed with trk receptors, p75NTR functionally collaborates with the trks to either enhance responses to preferred trk ligands, to reduce neurotrophin-mediated trk receptor activation resulting from non-preferred ligands or to facilitate apoptosis resulting from neurotrophin withdrawal. In cells lacking trk expression, p75NTR can act autonomously to activate ligand-dependent signaling cascades that may in some circumstances result in apoptosis but probably not through pathways utilized by its apoptotic brethren in the TNF receptor superfamily. Potential mechanisms for each of these functions of p75NTR are considered and the physiological implications of this unique signaling system are discussed.

Co-expression of the P75 neurotrophin receptor and neurotrophin receptor-interacting melanoma antigen homolog in the mature rat brain.

The p75 neurotrophin receptor (p75(NTR)) is involved in the regulation of neuronal survival and phenotype, but its signal transduction mechanisms are poorly understood. Recent evidence has implicated the cytoplasmic protein NRAGE (neurotrophin receptor-interacting MAGE (from Melanoma AntiGEn) homolog) in p75(NTR) signaling. To gain further insight into the role of NRAGE, we investigated the co-expression of NRAGE and p75(NTR) in mature rat brain. In all areas examined, NRAGE appeared to be confined to neurons. In the basal forebrain cholinergic complex, NRAGE immunoreactivity was evident in all p75(NTR)-positive neurons. There were many more NRAGE-positive than p75(NTR)-positive neurons in these regions, however. NRAGE was also expressed in areas of the basal forebrain that did not express p75(NTR), including the lateral septal nucleus and the nucleus accumbens. A finding in marked contrast to previous studies was the presence of p75(NTR) immunoreactivity in neuronal cell bodies in the hippocampus. Hippocampal p75(NTR) immunoreactivity was apparent in rats 6 months and older, and was localized to the dentate gyrus and stratum oriens. All p75(NTR)-positive neurons in the dentate gyrus and hippocampal formation were positive for NRAGE. The majority of granular cells of the dentate gyrus and pyramidal cells in the hippocampal formation were positive for NRAGE and negative for p75(NTR). NRAGE was also present in some neuronal populations that express p75(NTR) after injury, including striatal cholinergic interneurons, and motor neurons. A region of marked disparity was the cerebral cortex, in which NRAGE immunoreactivity was widespread whereas p75(NTR) was absent. The results are consistent with an important role for NRAGE in p75(NTR) signaling, as all cells that expressed p75(NTR) also expressed NRAGE. The wider distribution of NRAGE expression suggests that NRAGE may also participate in other signaling processes.

Lack of correlation between thyroid size and body growth in an area of endemic goiter.

There is conflicting information in the literature about the relationship between thyroid size and body growth in endemic goiter. This study reports data from highland Bolivia where 408 children were examined for thyroid size, height, weight, head circumference, and academic performance, the latter as measured by teacher evaluation. The presence or size of goiter showed no consistent correlation with height, weight, or head circumference. School performance was significantly correlated with all other variables in boys and not in girls, but there are severe limitations involved in the use of school grades alone for assessment of intelligence. Height and weight were less than for normals in the United States at each age level, but did not differ from those of other Quechuan children in neighboring Peru who were reportedly nongoitrous. Our data support the conclusion that there is no clear relationship between thyroid size and body growth in areas of endemic goiter.

The effects of oral iodized oil on intelligence, thyroid status, and somatic growth in school-age children from an area of endemic goiter.

One hundred goitrous school children received 475 mg iodized oil by mouth, while 100 controls received mineral oil, on a double-blind basis. On follow-up 22 months later the urinary iodine had increased and goiter size had decreased in both groups, more strikingly in the iodine-treated children. There were no consistent differences between the two treatment groups in rate of somatic growth or performance on the Stanford-Binet and Bender tests. Because of the complexities introduced by increases in urinary iodine in the controls, we compared goiter reduction with improvement in IQ score in all children, regardless of group, and found a significant relationship (p = 0.014), particularly in girls (p = 0.029). We conclude that oral iodized oil is an attractive alternative to its injection but we recommend an approximate doubling of the dose used here for more effective control. Also, while our data are not conclusive, they support the possibility that correction of iodine deficiency may improve mental performance in school age children, particularly girls.

Blocking nerve growth factor binding to the p75 neurotrophin receptor on sympathetic neurons transiently reduces trkA activation but does not affect neuronal survival.

Nerve growth factor interacts with the trkA tyrosine kinase receptor and with the p75 neurotrophin receptor. It is clear that trkA mediates most, if not all, of the stereotypical responses of sympathetic neurons to nerve growth factor but the role of the p75 neurotrophin receptor is unclear. In this study, we have asked whether a functional interaction between p75 neurotrophin receptor and trkA exists in primary sympathetic neurons by disrupting nerve growth factor binding to p75 neurotrophin receptor. Acute assays reveal that blocking antibodies directed against p75 neurotrophin receptor reduce nerve growth factor-mediated trkA tyrosine phosphorylation and reduce the amount of nerve growth factor which binds the trkA receptor. This reduction in trkA activity is relatively short-lived in vitro and blocking antibodies to p75 neurotrophin receptor do not inhibit long-term survival of nerve growth factor-dependent primary neurons. Together, these data indicate that p75 neurotrophin receptor and trkA interact within primary neurons to enhance nerve growth factor binding to the trkA receptor under conditions of acute but not chronic nerve growth factor exposure.

High frequency variability of environmental drivers determining benthic community dynamics in headwater streams.

Headwater streams are an important feature of the landscape, with their diversity in structure and associated ecological function providing a potential natural buffer against downstream nutrient export. Phytobenthic communities, dominated in many headwaters by diatoms, must respond to physical and chemical parameters that can vary in magnitude within hours, whereas the ecological regeneration times are much longer. How diatom communities develop in the fluctuating, dynamic environments characteristic of headwaters is poorly understood. Deployment of near-continuous monitoring technology in sub-catchments of the River Eden, NW England, provides the opportunity for measurement of temporal variability in stream discharge and nutrient resource supply to benthic communities, as represented by monthly diatom samples collected over two years. Our data suggest that the diatom communities and the derived Trophic Diatom Index, best reflect stream discharge conditions over the preceding 18-21 days and Total Phosphorus concentrations over a wider antecedent window of 7-21 days. This is one of the first quantitative assessments of long-term diatom community development in response to continuously-measured stream nutrient concentration and discharge fluctuations. The data reveal the sensitivity of these headwater communities to mean conditions prior to sampling, with flow as the dominant variable. With sufficient understanding of the role of antecedent conditions, these methods can be used to inform interpretation of monitoring data, including those collected under the European Water Framework Directive and related mitigation efforts.

Critical roles for the netrin receptor deleted in colorectal cancer in dopaminergic neuronal precursor migration, axon guidance, and axon arborization.

DCC (deleted in colorectal cancer), a receptor for the axon guidance cue netrin-1, is highly expressed by mesencephalic dopaminergic (DA) neurons during development; however, the contribution of DCC to DA development remains largely uncharacterized. DA neurons in ventral mesencephalic nuclei also express UNC5 homologue netrin receptors from late embryogenesis to adulthood, raising the possibility that DA axons could be attracted or repelled by netrins. Examining newborn dcc null mice, we report that loss of DCC function results in profound alterations of DA circuitry, including DA progenitor cell migration defects, reduced numbers of DA cells in midbrain nuclei, an anomalous DA ventral commissure, malformed DA innervation of the ventral striatum, and reduced DA innervation of the cerebral cortex. Caspase-3 activation was detected in inappropriately localized DA cells, consistent with apoptosis contributing to reduced cell numbers. Dcc heterozygous mice express reduced levels of DCC protein. Although less severely disrupted than dcc nulls, newborn and adult dcc heterozygotes also have fewer DA neurons in ventral mesenscephalic nuclei. Despite the reduced numbers of DA neurons, newborn dcc heterozygotes and nulls exhibit similar DA innervation density as wild-type littermates in the nucleus accumbens core, and adult dcc heterozygotes exhibit increased DA innervation in medial prefrontal cortex. A trend towards increased innervation of medial prefrontal cortex was detected in newborn dcc heterozygotes, but did not reach statistical significance, suggesting that the increase in adult heterozygotes results from enhanced DA arborization during postnatal development. Consistent with the hypothesis that DCC regulates DA axonal projections, disrupting DCC function in culture inhibits netrin-1 induced DA axon extension and axon branching. Furthermore, disrupting DCC function in isolated DA neurons grown as micro-island cultures reduces the number of autaptic synapses per cell. We conclude that DCC regulates appropriate precursor cell migration, axon guidance, and terminal arborization by DA neurons.

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo.

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.

TTIP is a novel protein that interacts with the truncated T1 TrkB neurotrophin receptor.

Alternative splicing of the TrkB gene produces a full length tyrosine kinase receptor as well as two truncated isoforms that contain extracellular and transmembrane domains but lack the kinase domain and have unique C terminal tails. The function of the truncated TrkB isoforms is unclear and to gain insights into their function, we have isolated a protein from 15N neuroblastoma cells that specifically binds the TrkB.T1 isoform. Pulldown experiments using a GST fusion protein containing the TrkB.T1 intracellular domain identified a 61 kDa protein from radiolabeled 15N lysates. Coimmunoprecipitation experiments showed that the 61 kDa protein interacted with epitope-tagged TrkB.T1 overexpressed in 15N cells as well as with TrkB.T1 which was endogenously expressed. Peptide competition experiments revealed that the protein, designated TTIP (for Truncated TrkB Interacting Protein), showed specific binding to the TrkB.T1 tail. MALDI MS and MS/MS analysis has revealed that TTIP is a novel protein not yet listed in the current databases.