bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevention of apoptosis and preservation of function by protease inhibitors in cortical neurons exposed to glutamate
Authors: Ray S. K. , Nowak M. W. , Li L. , Wilford G. G. , Banik N. L.
Published: July 2010

Traumatic injury and diseases of the central nervous system are often associated with release of L‐glutamate that causes neuronal death and dysfunction. Rat primary cortical neurons exposed to 0.5 μmL‐glutamate for 24 h died mostly by apoptosis (Wright staining, ApopTag assay). L‐glutamate exposure also caused up‐regulation of calpain and caspase‐3 compared with β‐actin, an internal control (Western blot). We tested calpain and caspase‐3 inhibitors to prevent death and dysfunction in following L‐glutamate exposure. Pre‐treatment of cells with 0.2 μm calpeptin (calpain‐specific inhibitor) or/and 100 μm z‐DEVD‐fmk (caspase 3‐specific inhibitor) prevented apoptosis. Electrophysiological properties and whole‐cell currents associated with voltage‐gated Na+ channels, NMDARs, and kainate/AMPARs were measured. There were no significant differences in VREST, membrane capacitance and IBASAL at −70 mV for inhibitor only or inhibitor plus L‐glutamate treated neurons relative to control neurons. The lack of a change in capacitance indicated that neurons treated with inhibitor plus L‐glutamate did not undergo apoptotic shrinkage and maintained the same size as the control neurons. Currents associated with Na+ channels, NMDARs, and kainate/AMPARs were similar in amplitude and activation/inactivation kinetics for control and treatments with inhibitor plus L‐glutamate. Spontaneous synaptic activity (as observed by miniature end‐plate currents) was also similar in control, inhibitor only, and inhibitor plus L‐glutamate treated cultures. Prevention of L‐glutamate‐induced apoptosis by protease inhibitors preserved normal electrophysiological properties and ion channel activity in cortical neurons.

Acknowledgements:  Supported by the grants from NIH‐NINDS (NS31622, NS38146, NS41088).
Poster Sessions AP01: Gene Expression and Regulation. Regulation of gene expression during the development of cortico‐thalamic connection in rat
Authors: Calandria J. M. , Alfonso J. , Pollevick G. , Casas O. R. , Frasch C. , Idoyaga‐Vargas V.
Published: July 2010

The formation of Cortico‐Thalamic projections requires the precise spatial and temporal expression of proteins that are involved in the different stages of synaptogenesis. We reasoned that the underlying molecular mechanism of this process is the differential expression of genes that code for stage specific proteins. Our research objective was to identify the differential expressed mRNAs during the main stages of synapses formation, which starts at embryonic day 12 (E12) and finishes on the first postnatal days in the rat. We approach this problem using Differential Display technique on three distinct ages of rat cerebral cortex that were: E13, E18 and postnatal day 0 (P0). We found 80 differential bands using 54 random primers and 18 of them were cloned and sequenced. The sequence analysis showed among others, a cDNA fragment highly homologous with the human A Kinase Anchoring Protein 450/350 also called CG‐NAP. We found that this cDNA fragment homologous to AKAP was up regulated at E15 when cortical cells are undergoing active axogenesis. The expression pattern of this cDNA was confirmed by Real Time PCR. Our findings suggest a possible function for AKAP 450 in the regulation of the state of phosphorylation of centrosomal components during the initial stages of synapses formation during the establishment of Cortico‐Thalamic connection.
The MyT1 family recruits histone deacetylase to regulate neural transcription
Authors: Romm E. , Kim J. G. , Kim N. W. , Nagle J. , Hudson L. D.
Published: July 2010

The MyT1 gene family is comprised of three zinc finger genes (MyT1, MyT1‐Like/MyT1L and NZF3) of the structurally unique CCHC class that are expressed predominantly in the developing central nervous system. To understand the molecular mechanism by which this family promotes neural differentiation, we searched for interaction partners. In both a yeast and a mammalian two hybrid system, MyT1 and MyT1L interacted with Sin3B, a protein, which can mediate transcriptional repression by binding to histone deacetylase (HDAC). MyT1‐Sin3B complexes could also be coimmunoprecipitated from transfected mammalian cells. The interacting domain includes three amphipathic alpha‐helices and is highly conserved among MyT1 family members. MyT1 and MyT1L can partner with both Sin3B isoforms, the full length form that includes the HDAC‐binding domain and an alternatively spliced short form (Sin3BSF) that lacks this domain and would consequently antagonize Sin3B‐mediated corepression. MyT1 has two zinc finger clusters, and one of these DNA‐binding domains recognizes a broad consensus site (GGTGGGGa/g a/g a/g) suggestive of a wide range of potential gene targets for this transcription factor. We present a model in which the MyT1 family of zinc finger proteins, when tethered to the DNA binding site of a neural promoter, can recruit the corepressor Sin3B. Depending on the relative availability of Sin3B isoforms, the MyT1 gene family may favor the silencing or activation of selected genes during neural development.
P2X7 receptor induces gene expression
Authors: Santos‐Berríos C. , Weisman G. A. , Gonzalez F. A.
Published: July 2010

The concentration of extracellular nucleotides is known to rise significantly under inflammatory conditions in vivo. In normal tissues, extracellular nucleotides are maintained at low concentrations and tightly regulated. However, activated lymphocytes, macrophages, microglia, and platelets, as well as cells undergoing necrosis or apoptosis, release high concentrations of different nucleotide diphosphates and triphosphates into the extracellular space. It has also been shown that P2 nucleotide receptors ligands modulate IL‐1β‐induced transcription factor activation and differentially regulate inflammatory gene expression in primary human astrocytes. In previous work using cDNA microarray technology and RT–PCR, we found that chronic exposure of human monocytic U937 cells to UTP modulated the expression of a large number of genes related to the monocyte to macrophage differentiation process, including the tumor necrosis factor (TNF‐α). Here we present the effect of chronic exposure to extracellular nucleotides and nucleotide analogs on the expression of genes in 1321 N1 astrocytoma cells expressing recombinant P2X7 receptors. Our findings will help us better understand the role of nucleotide receptors in the inflammatory response in the nervous system, information that is bound to have pharmacological applications and clinical significance.
Copper and amyloid β: interactions in Alzheimer's disease
Authors: Yusof M. , Sun A.
Published: July 2010

As the leading cause of dementia, Alzheimer's Disease (AD) is characterized by a loss of memory and neurons. These characteristics have been associated with brain lesions known as neurofibrillary tangles and amyloid plaques, which consist of 39–42 amino acid amyloid beta peptides (Aβ). The exact nature of the pathogenesis of amyloid toxicity in AD is yet to be determined, however, it is likely that highly susceptible neurons are responding to oxidative stress via mechanisms that may promote the tangle and plaque formation. The amyloid precursor protein and Aβ have been shown to have binding sites for copper, a highly reactive redox metal. The current study is of the effects of copper on Aβ neurotoxicity using rat pheochromocytoma cells and rat primary neurons. The data show that Aβ induced cell death and that this is further potentiated by copper in physiologically relevant concentrations (50 μm). Moreover, copper chelator and antioxidant therapy show protection of the cell damage. Measurements of cell damage include cell viability and lipid peroxidation studies. Collectively, the results indicate that copper may play an important role in the neurotoxicity of Aβ, cell death and the formation of amyloid plaques in AD patients.
A new method for measuring fibrillar amyloid β‐protein using diphenylhexatriene, a fluorescent probe
Authors: Chauhan A. , Chauhan V.
Published: July 2010

The two current methods for the measurement of fibrillar (f) amyloid beta‐protein (Aβ), namely Thioflavin T (ThT) fluorescence spectroscopy and Congo red (CR) absorbance spectroscopy, are based on the interaction of fAβ with cationic ThT and anionic CR molecules. The presence of cations and colored factors in the reaction often interferes with ThT and CR assays. Recently, we reported that fibrillar Aβ forms a membrane‐like hydrophobic domain (Chauhan et al. 2001; Neuroreport12, 587–590). We report here that diphenylhexatriene (DPH), a neutral fluorescent probe, interacts with fibrillar Aβ 1‐40 and fibrillar Aβ 1‐42, but not with soluble Aβ. As a result, DPH fluorescence increases several fold in the presence of fibrillar Aβ at the excitation and emission wavelength of 360 and 430 nm, respectively. The blank sample (DPH in buffer) had minimal fluorescence, and soluble Aβ had no effect on DPH fluorescence. The interaction of DPH with fibrillar Aβ was dependent on the incubation time of these two constituents. At fixed Aβ concentration and varying DPH concentration, there was a lag phase of DPH fluorescence from 1 to 4 μm DPH, that was followed by a linear increase in fluorescence from 4 to 8 μm DPH which then reached a plateau. Studies at varying pH showed that emission spectra of fAβ/DPH can be studied at a wide range of pH from 4 to 8. Aβ concentration studies showed that the fluorescence of DPH increases linearly with increase in the concentration of fibrillar Aβ 1‐40/Aβ 1‐42. Kinetic studies showed that one mole of fAβ binds to 15.5 mol of DPH. These results suggest that DPH fluorescence can be used as a standard assay method for the measurement of fibrillar Aβ.
Perturbed sphingomyelin metabolism and accumulation of ceramides and cholesterol esters in Alzheimer's disease and ALS
Authors: Mattson M. P. , Cutler R. G. , Pedersen W. A.
Published: July 2010

Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) involve selective degeneration of neurons involved in learning and memory (hippocampal and cortical neurons) and movement (spinal cord motor neurons), respectively. In each disorder and increase in oxidative stress is believed to render neurons vulnerable to excitotoxicity and apoptosis. We report abnormalities in sphingolipid and cholesterol metabolism in brain tissue of AD patients and a mouse model of AD (APP mutant mice), and in spinal cords of ALS patients and a transgenic mouse model of ALS (Cu/Zn‐SOD mutant mice), characterized by increased levels of sphingomyelin, long‐chain ceramides and cholesterol esters. In the mouse models these abnormalities precede the clinical phenotype. Exposure of cultured hippocampal neurons to amyloid β‐peptide (the neurotoxic component of amyloid plaques in AD) and of cultured motor neurons to oxidative insults, increases the accumulation of sphingomyelin, ceramides and cholesterol esters. Pharmacological inhibition of sphingomyelin synthesis prevents accumulation of ceramides and cholesterol esters and protects hippocampal and motor neurons against death induced by insults relevant to AD and ALS. These findings suggest links between oxidative stress, altered sphingolipid and cholesterol metabolism and the pathogenesis of AD and ALS. Our data also identify novel targets for therapeutic intervention in neurodegenerative disorders.
Molecular targeting of Alzheimer's amyloid plaques for contrast‐enhanced magnetic resonance imaging
Authors: Poduslo J. F. , Wengenack T. M. , Curran G. L. , Wisniewski T. , Sigurdsson E. M. , Macura S. I. , Borowski B. J. , Jack C. R.
Published: July 2010

Smart molecular probes for both diagnostic and therapeutic purposes are expected to provide quantum advances in clinical medicine and biomedical research. We describe such a probe that targets amyloid plaques of Alzheimer's disease and has been labelled with gadolinium to impart contrast detectable by magnetic resonance imaging. Although amyloid plaques are a primary pathologic feature of Alzheimer's disease and amyloid reduction is a major therapeutic objective in Alzheimer's disease, no method currently exists for directly imaging human amyloid plaques in vivo. However, with the aid of a target specific contrast agent, imaging of amyloid plaques is theoretically possible with magnetic resonance imaging. We describe experiments proving that three essential properties exist in the smart molecular probe, putrescine‐gadolinium‐AB peptide: (1) transport across the blood–brain barrier following intravenous injection conferred by the polyamine moiety (2) binding to plaques with high affinity by putrescine‐AB, and (3) magnetic resonance imaging contrast by gadolinium. The plaque‐to‐background tissue contrast‐to‐noise ratio was enhanced more than 9 fold in all regions of cortex and hippocampus following intravenous administration of putrescine‐gadolinium‐AB in Alzheimer's disease transgenic mice. The ability of this magnetic resonance contrast probe to image plaques in vivo may enable early diagnosis and also provide a direct measure of the efficacy of antiamyloid therapies currently being developed. With the appropriate target specific probe (e.g. antibody), this technique could be applied to many other central nervous system disorders and targets.
A role for the transcription factor ets‐2 in Down's syndrome neuropathology
Authors: Pelsman A. , Busciglio J.
Published: July 2010

Down's Syndrome (DS) is the most frequent genetic cause of mental retardation. Most DS patients develop Alzheimer's disease by middle age. A transgenic model of ets‐2 that presents pathological features similar to DS suggests a role for ets‐2 in DS (Sumarsono et al. 1996; Nature379, 534). We analyzed the expression and activity of ets‐2 in normal and DS brain tissue and human cortical neurons (HCN) in culture. Western blot and immunocytochemistry revealed that ets‐2 is expressed in the human brain and in HCN during development. The level of ets‐2 was significantly increased in DS brain homogenates and DS HCN cultures. Interestingly, DS HCN cultures exhibited reduced bcl‐2 and increased bax and p53 protein levels consistent with reduced DS neuronal viability in culture. To determine the role of ets‐2 in neuronal degeneration, normal neurons were transfected with an ets‐2 expression vector. After 24 h, a significant increase in cell death was observed in ets‐2‐transfected neurons but not in neurons cotransfected with a dominant‐negative ets‐2 expression vector (ets‐2dn). Neuronal degeneration was dependent on p53 activity since a dominant‐negative mutant form of p53 significantly abolished ets‐2‐induced neuronal degeneration. Moreover, ets‐2 overexpression did not increase neuronal death in neuronal cultures derived from p53KO mice. Finally, the expression of ets‐2dn significantly reduced degeneration of DS cortical neurons in culture. In DS, ets‐2 overexpression may compromise neuronal viability directly by inducing neuronal apoptosis or indirectly by rendering neurons more sensitive to pro‐apoptotic stimuli such as Aβ neurotoxicity and oxidative stress.
CST1 antibody specifically recognizes cold stable tubulin
Authors: Wray R. , Szebenyi G. , Brady S. T.
Published: July 2010

Cold stable tubulin (CST) is isolated by a cold calcium fractionation method that results in a cold soluble fraction and a stable cold insoluble fraction. Previous biochemical studies suggest that a post‐translational modification, polyamination, is responsible for the stable form of microtubules. CST is enriched in the adult central nervous system. However, because of a mixed population of cells in biochemical studies, little is known about CST's cellular distribution. Without an antibody against CST, the cellular localization and tissue distribution could not be determined definitively. Therefore, we developed a monoclonal antibody to CST. The antibody was raised and screened against the gel purified cold insoluble fraction. Its specificity for CST was then tested using ELISA, Western blotting and immunostaining. One clone (CST1) specifically recognized the cold insoluble but not the cold soluble microtubule fraction on both Western blot and ELISA. With immunostaining in the adult rat brain, reactivity was seen in both cortical neurons and axon tracks. The staining was restricted to a subset of NF‐L positive neurons. The signal in projection neurons was enriched in cell bodies and proximal axons, but was not detected in MAP2 positive dendrites. With this novel probe we can now gain a better understanding of CST's distribution and function in the CNS.
Insulin‐like signalling in neurons controls lifespan in C. elegans
Authors: Wolkow C. A.
Published: July 2010

Insulin‐like signalling controls C. elegans lifespan, development and metabolism. Mutations that weaken this insulin‐like signalling pathway extend lifespan. Severe mutations abolishing insulin‐like signalling cause animals to arrest development as dauer larvae, a larval form specialized for stress resistance and long‐term survival. A number of the genes acting in this pathway have been cloned, including daf‐2, which encodes a homolog of vertebrate insulin/IGF‐I receptors, and age‐1, encoding the C. elegans homolog of the PI(3)K p110 catalytic subunit. In order to identify cells from which insulin‐like signalling controls lifespan and development, transgenic animals were constructed which possessed insulin‐like signalling only in specific cell types. To achieve this, cell‐type specific promoters were used to drive expression of daf‐2 or age‐1 cDNAs in daf‐2(–/–) or age‐1(–/–) backgrounds, respectively. By utilizing this strategy, we could restore wild‐type daf‐2 or age‐1 activity only in cells that are capable of expressing each transgene. Restoring insulin‐like signalling to the nervous system of daf‐2 or age‐1 mutants could rescue long lifespan. This result was specific for transgenes restoring insulin‐like signalling to the nervous system. Expressing daf‐2 or age‐1 cDNAs from muscle‐ or intestinally‐restricted promoters was insufficient to rescue lifespan. In contrast, age‐1 and daf‐2 expression in either neuronal or non‐neuronal cell types rescued dauer larval arrest in the mutants. These findings demonstrate that insulin‐like signalling pathways in the nervous system control C. elegans lifespan.
Retinoic acid's role in sexually dimorphic neural homeosis
Authors: Rosa‐Molinar Eduardo
Published: July 2010

The sexually dimorphic developmental program of the western mosquitofish, Gambusia affinis affinis radically reorganizes the typical two compartment teleost body plan and nervous system (e.g. pre‐caudal: vertebral bodies with ribs; and caudal: vertebral bodies without ribs and no ano‐urogenital plexus) into a three compartment body plan [e.g. pre‐caudal: vertebral bodies with ribs; and caudal: vertebral bodies without ribs and an ano‐urogenital region (a region composed of pre‐caudal and caudal vertebrae combined)] and a ano‐urogenital plexus and is reminiscent of homeotic transformations achieved by disturbing developmental programs using exogenous retinoic acid (RA). RA has not been thought to play a role in the patterning, organization and regulation of neural sexual dimorphism, a role considered to be the domain of steroids, specifically, testosterone and estrogen. Data discussed in this presentation will show the cell types responsible for synthesizing RA within the spinal cord of G. a. affinis. We will present data that the endogenous RA synthesized within the spinal cord is an instructive morphogen that (1) imparts identity and position of sex‐specific vertebral and motor neurons and (2) regulates the number of motor neurons within the spinal cord region associated with the ano‐urogenital region. These findings may represent the first described example of ‘sexually dimorphic neural homeosis’.

Acknowledgements:  Research support through a grant from the National Science Foundation (IBN‐0091120).
Poster Sessions CP03: Trophic Factors. Effects of early post‐natal exposure of cerebral cortex to hyper‐IL‐6 on development and cognition in the CD‐1 mouse
Authors: Brunssen S. H. , Houser E. R. , Harry G. J.
Published: July 2010

Pro‐inflammatory cytokines, especially Interleukin (IL)‐6, are elevated in the brains and serum of very premature infants in association with perinatal infectious and hypoxic/ischemic insults. To determine potential adverse effects of IL‐6 on brain development, cross‐fostered litters of male cd‐1 mice were injected subcranially with hyper‐IL‐6 or vehicle at postnatal day (pnd) 4 and examined for alterations in functional landmarks of CNS development. No acute injury response was evident at 1–7 days following hyper‐IL‐6 injection as determined by glial morphology and RNase protection assays for acute response genes, ICAM‐1, iNOS, Mac‐1, A‐20, and EB‐22. In preweanling mice, reflex ontogeny and physical markers of development were not altered; however, exposed mice demonstrated increased over‐generalized motor responses (Fox Battery) leading to an overall impression of hyper‐reactivity and hyperactivity. At pnd 24 and 80, assessment of autonomic, sensory, and motor systems (Functional Observation Battery) showed persistence of hyper‐reactivity and hyperactivity. Learning and memory were assessed by passive avoidance (PA) at pnd 24 and by radial arm maze (RAM) in adults. Hyper‐IL‐6 exposure significantly impaired PA performance. RAM performance was similar between hyper‐IL‐6 and control groups in acquisition of win‐shift task but hyper‐IL‐6 animals showed deficits on repeated acquisition task. The persistent hyperactive, hyper‐reactive behavioral phenotype evident in hyper‐IL‐6 exposed mice appeared to interfere with performance on both learning and memory tasks, suggesting that early exposure of the developing brain to IL‐6 can have persistent adverse functional effects and raising additional concerns for effects of immune mediators in premature infants.
Gangliosides activate Trk receptors by inducing the release of neurotrophins
Authors: Mocchetti I. , Bachis A. , Rabin S. J.
Published: July 2010

We used NIH‐3T3 fibroblasts expressing the different Trk receptors to examine whether GM1 ganglioside and its semisynthetic derivative LIGA20 activate various neurotrophin receptors. GM1 elicited a rapid activation of TrkC and TrkA, but not TrkB, tyrosine phosphorylation. In contrast, LIGA20 activated TrkB tyrosine phosphorylation only. These data suggest that gangliosides may act as ligands for specific Trk receptors similar to the neurotrophins. To test this hypothesis, we performed Scatchard analysis to determine if GM1 binds to the TrkC receptor. GM1 failed to displace neurotrophin‐3 (NT‐3) binding suggesting that gangliosides are not ligands for the Trk receptors. Moreover, GM1 did not activate a TNF/Trk chimeric receptor. We next determined whether gangliosides induce the release of neurotrophins. NIH‐3T3 cells and cerebellar granule neurons were exposed to GM1 or LIGA20 for 5 min and the levels of brain‐derived neurotrophic factor (BDNF) and NT‐3 measured in the medium. GM1 induced a significant increase in the amount of NT‐3, but not BDNF. In contrast, LIGA20 increased the secretion of BDNF, suggesting that gangliosides differentially affect the release of neurotrophins, which, in turn, activate Trk receptors. Therefore, Trk tyrosine phosphorylation was determined in the presence of Trk‐IgG receptor bodies. TrkB‐IgG, but not TrkC‐IgG, inhibited LIGA20‐mediated induction of TrkB tyrosine phosphorylation, while TrkC‐IgG, but not TrkB‐IgG, inhibited GM1‐mediated activation of TrkC, further suggesting that gangliosides activate Trk receptors by inducing the release of endogenous neurotrophins.
Oligodendrocyte vesicle transport: role of Rab40c
Authors: Larocca J. N. , Almazan G. , Rodriguez‐Gabin A. G.
Published: July 2010

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin and regulation is necessary to maintain the structural and functional organization of the oligodendrocytes. Exocytic events are involved in the targeting of proteins synthesized in the endoplasmic reticulum such as proteolipid protein, DM20 and MAGs. Endocytic events also play an important part in the formation and maintenance of myelin as indicated by endocytosis in oligodendrocyte processes and myelin domains. Rab proteins are key components in mechanisms that regulate intracellular membrane trafficking. We cloned a novel oligodendrocyte Rab protein, designated Rab40c from its homology with Rab40. The cDNA sequence of Rab40c and the deduced amino acid sequence show an 843 base open reading frame indicating a protein with 281 amino acids. This protein has a deduced molecular of weight 31 466 Da and an isoelectric point of 9.83. Rab40c presents a number of distinct structural features including a carboxyl terminal extension and the occurrence of amino acid substitutions in the consensus sequence of the GTP‐binding motifs. The carboxyl terminal region contains motifs that indicated possible isoprenylation and palmitoylation. Rab40c is localized in the perinuclear endocytic recycling compartment suggesting that it is involved in the regulation of endocytic events such as the recycling of receptors. The importance of this recycling in myelin formation is indicated by the increase in Rab40c mRNA with differentiation of oligodendrocytes.
Endothelin‐B receptor mediates effect of intravitreal endothelin on mitochondria‐associated anterograde axonal transport
Authors: Stokely M. E. , Brady S. T. , Yorio T.
Published: July 2010

Increased endothelins (ETs) are associated with glaucoma and have been proposed to contribute to development of glaucomatous optic neuropathy. In glaucoma, movement of anterogradely transported material important for ganglion cell survival is impaired, specifically transport of mitochondria. This study characterizes effect(s) of a single bolus of intravitreal endothelin (nonselective ET‐1, or ETb‐receptor selective ET‐3) on the mitochondrial subcomponent of anterograde axonal transport. Standard segmental analysis techniques were used to pulse‐label and analyze newly synthesized proteins undergoing anterograde axonal transport in optic nerves of male Sprague–Dawley rats (n = 7; 35S‐methionine ± 2 nmols ET‐1 or ET‐3). Injection‐sacrifice intervals (ISIs) of 24, 28, 32, & 36 h were evaluated (anova). Effects of intravitreal ET‐1 are biphasic with maximal impact at times normally associated with mitochondrial protein transport (28–36 h). Total newly synthesized protein in the mitochondrial subcomponent was diminished by both ET‐1 treatment (28, 32, 36 h ISIs; ps = <0.001, 0.02, <0.001, respectively) and ET‐3 treatment (28 h, p < 0.01), with no difference between effects of ET‐1 and ET‐3 at 28 h (p < 0.999). Data from 11 SDS–PAGE protein bands (139, 118, 89, 80, 64, 59, 51, 45, 42, 37, 25 kDa) in the mitochondrial subcomponent, indicate that ET‐1 both diminished (all p's < 0.05) the amount of protein transported and consistently delayed appearance of labelled protein in the optic nerve by 4 h. These results are consistent with an ETB‐receptor mediated role for endothelins in the pathologic dysregulation of mitochondria‐associated anterograde axonal transport.
Poster Sessions CP08: Signal Transduction. Synergistic modification of inducible and neuronal NOS expression by NGF and TNFα: relevance to CNS aging
Authors: Taglialatela G. , Thomas M. S. , Zhang W. R. , Macdonald N. J. , Andorn A. C.
Published: July 2010

Inducible nitric oxide synthase (iNOS) and high levels of nitric oxide (NO) are present in the CNS of patients with Alzheimer's disease (AD), resulting in both DNA and protein oxidative damage. While iNOS can result in damaging levels of NO, the neuronal constitutive form of NOS (nNOS) has a role in cell signalling and can prevent neuronal apoptosis. iNOS can be induced by inflammatory cytokines such as tumor necrosis alpha (TNFα). TNFα is found in the CNS of AD, where neurons dependent on neurotrophins such as nerve growth factor (NGF) are particularly affected. Here we determined the effect of TNFα on the three NOS isoforms (endothelial, neuronal and inducible) in NGF‐responsive PC12 cells. We found that while TNFα and NGF alone were uneffective, their simultaneous addition resulted in iNOS induction and the release of NO. In addition TNFα and NGF synergistically reduced nNOS, independently of the presence of high NO levels promoted by iNOS, while no effect was observed on eNOS. A similar pattern was observed in the brain of aged human subjects as compared to young individuals. Our results suggest that synergistic iNOS induction by TNFα and NGF may occur in selective populations of NGF‐responsive neurons. Oxidative damage in such neurons could then occur in the presence of elevated levels of TNFα, that potentially occur in the brain of AD patients. This damaging scenario may further be aggravated by a concomitant reduction of nNOS, brought about by similar synergistic effects between TNFα and NGF.

Acknowledgements:  Supported by NIA (AG13945) and Sealy Res. Dev. grants to GT.
Membrane lipid regulation of the tumor suppressor phosphatase PTEN
Authors: Goswami R. , Singh D. , Phillips G. , Kilkus J. , Dawson G.
Published: July 2010

The neutral sphingolipid ceramide has been implicated in the apoptotic death of cells by a number of different mechanisms and we now present evidence that one of its actions is to activate the tumor supressor protein PTEN (phosphatase and tensin homolog deleted from chromosome 10). PTEN is mutated in multiple advanced cancers, is absent from many tumor cells such as gliomas, and functions largely as a polyphosphoinositide phosphatase. PTEN with a red‐fluorescent protein (RFP) tag was overexpressed in both a human cell line derived from oligodendroglioma (HOG) and a rat pheochromocytoma cells line (PC12) by means of an inducible promoter system (tet‐OFF). Induction of the gene by removal of doxocycline showed a generalized (cytosolic) distribution of PTEN which was changed to a more membrane‐associated pattern following treatment with C2‐ceramide. Activation of the cell death pathway with either staurosporine, wortmannin or C2‐ceramide was facilitated by PTEN expression while lowering PTEN expression with antisense constructs partially blocked cell death. The staurosporine‐induced increase in ceramide was localized to the Raft (detergent‐resistant‐membrane fraction) where active PTEN is also found. We propose that formation of ceramide during apoptosis results in the translocation of PTEN to the plasma membrane, where it can hydrolyze the polyphosphoinositides which normally activate the key antiapoptotic protein, phospho‐Akt (protein kinase B). Since ceramide may also activate a Phospho‐Akt phosphatase, this may further explain the high pro‐apoptoic potential of ceramide.
Activation of aromatic l‐amino acid decarboxylase by calcium/calmodulin kinase II
Authors: Neff N. H. , Duchemin A‐M. , Hadjiconstantinou Maria
Published: July 2010

Calcium/calmodulin‐dependent protein kinase II (CAMKII) is widely distributed in the brain, and is involved in the regulation of neurotransmitter synthesis. Tyrosine hydroxylase and tryptophan hydroxylase, the two enzymes involved in the first step in the synthesis of catecholamines and indolamines, respectively, are phosphorylated by CAMKII. We now found that aromatic l‐amino acid decarboxylase (AAAD), the common second enzyme of both synthetic pathways, is activated by CAMKII. AAAD activity was assayed in mouse striatum homogenates with L‐DOPA as a substrate. The activation of AAAD by CAMKII in vitro is time and concentration‐dependent with maximal activity observed with 1.5 μg/mL of purified brain CAMKII and a 20‐min incubation. CAMKII induces a 30% increase of the apparent Vmax of the enzyme without changing its affinity for L‐DOPA and the cofactor pyridoxal‐5′‐phosphate. We have previously shown that AAAD can be phosphorylated by cyclic AMP‐and cyclic GMP‐dependent protein kinases. These two kinases induced an increase of AAAD activity of about 20%. Phosphorylation by several differentially regulated serine‐threonine kinases might be the mechanism responsible for the short‐term kinetic activation of the enzyme, which has been found in vivo after physiological stimuli and neurotransmitter receptor modulation.

Acknowledgements:  Supported, in part, by grant NS34571.
Lorenzo's oil, adrenoleukodystrophy, and the blood– brain barrier
Authors: Murphy E. J.
Published: July 2010

Adrenoleukodystrophy is a rapid, progressive demyelinating disease affecting the CNS that is characterized by large increases in plasma and tissue very long saturated fatty acids (VLCFA). Lorenzo's oil (LO), consisting of erucic (22:1 n‐9) and oleic (18:1 n‐9) acid in a triglyceride form, is a dietary therapy effective in reducing plasma and tissue VLCFA. Despite the decreased VLCFA, clinical studies indicated that LO failed to stop the progressive demyelination, suggesting that erucic acid, the active component of LO, did not cross the BBB. We addressed this question by infusing [14‐14C] 22:1 n‐9 (170 μCi/kg) into male rats using two different infusion paradigms. The radiotracer was infused (i.v.) into awake, adult male rats over a 10‐min period or infused (i.c.v.) into the fourth ventricle over a 7‐day period using an osmotic mini‐pump. Brains were removed from the cranium, frozen in liquid nitrogen, lipids extracted, and separated using standard techniques. [1‐14C] 20:4 n‐6 was infused (i.v.) and used as a positive control. Following i.v. infusion, 0.011% of the erucic acid was extracted by the brain, compared to 0.055% of the arachidonic acid. About 60% of the brain erucic acid was found in the aqueous fraction compared to 30% for arachidonic acid. Further, erucic acid was targeted to cholesteryl ester and triacylglyceride pools, whereas arachidonic acid was targeted to phospholipid pools. In animals infused i.c.v., 0.078% of the dose was taken up and about 60% of the erucic acid was targeted to phospholipid pools. These results clearly demonstrate that erucic acid crosses the BBB, similar to arachidonic acid, and is incorporated into specific lipid pools.

Acknowledgements:  This work was supported by The Myelin Project.
Hypothalamic ependymal cells express the glucose transporter GLUT2, a protein involved in glucose sensing
Authors: Nualart F. , García M. A. , Ulloa V. , Silva C. , Pastor P. , Vera J. C. , Reinicke K.
Published: July 2010

The GLUT2 glucose transporter is expressed in pancreatic islet β cells participating in glucose sensing mechanisms. In addition to glucose, GLUT2 is required in the transport of fructuose. In situ hybridization analyses of GLUT2 gene expression suggested that this transporter is present in hypothalamic regions responsible for glucose sensing. The hypothalamic glial cells, known as α and β tanycytes, are specialized ependymal cells that bridge the CSF and the blood. We used immunocytochemistry and transport analyses to demonstrate that GLUT1 and GLUT2 are expressed in tanycytes and are directly involved in the uptake of glucose and fructose. We also demonstrated that GLUT1 expression is elevated in β‐1 tanycytes, as compared to neurons and astrocytes. In addition, confocal microscopy analysis using anti‐GLUT2 indicated that GLUT2 was specifically expressed in β‐1 and β‐2 tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was found to express both GLUT1 and GLUT2. Analyses of 2‐deoxy‐d‐glucose and fructose uptake in cultured tanycytes revealed the presence of two transporters with transport Kms and sensitivity to inhibitors consistent with the functional properties of GLUT1 and GLUT2. Therefore, we conclude that mouse hypothalamic tanycytes express functional GLUT1 and GLUT2. Because the expression of GLUT2 is linked to glucose sensing mechanisms, we postulate that glial cells participate, at least in part, in glucose sensing mechanisms.

Acknowledgements:  Supported by Grands FONDECYT 1010843 and DIUC‐GIA 201.034.006–1.4 from Concepción University.
Gene expression for glycosyltransferases in transfected neuroblastoma cells
Authors: Irwin L. N. , Bieberich E. , Li D. D. , Silva J. , Yu R. K.
Published: July 2010

To study regulatory mechanisms of ganglioside biosynthesis, genes for sialyltransferase II (ST2) or acetylgalactosaminyltransferase I (GalNAcT) were transfected into different cell lines of a substrain of murine neuroblastoma F‐11 cells (F‐11A). While complex β‐series ganglioside synthesis is negligible in the parental line, cells transfected with genes for either enzyme express complex gangliosides requiring both enzyme activities. We have used primers specific for each of the two genes to determine the level of gene expression (1) in the parental cells where endogenous ST2 and GalNAcT activities are very low, and (2) in each of the transfected cell lines where both enzyme activities are present and complex gangliosides are synthesized. Transcripts for both ST2 and GalNAcT were amplified from cDNA that was reverse‐transcribed from the parental as well as both transfected cell lines. While some differences in transcript number and size were noted between parental, ST2‐transfected, and GalNAcT‐transfected cells, suggesting some degree of transcriptional control, all the cell lines clearly expressed transcripts for both genes. These results support the view that ST2 and GalNAcT act in a coordinated fashion, perhaps as a multienzyme complex, to catalyze the synthesis of complex gangliosides, and that synthesis of complex β‐series gangliosides in these cells is regulated primarily post‐transcriptionally.

Acknowledgements:  Supported by NIH R15‐DC05179 to L.N.I, NIH NS‐11853 to R.K.Y, and NIH‐NCRR(RCMI) G12‐RRO8124 to the Border Biomedical Research Center at the University of Texas at El Paso.
Prediction, isolation, and sequencing of the promoter for human aminophospholipid translocase (APTL) II
Authors: Sobocki T. , Sobocka M. B. , Banerjee P.
Published: July 2010

p‐Type ATPases drive translocation of phosphatidylserine (PS) and phosphatidyl‐ethanolamine (PE) from the outer to inner leaflet of the plasma membrane. This results in their inner‐leaflet sequestration, which is typical for healthy cells. The externalization of PS (and PE) during apoptosis labels apoptotic cells for phagocytosis by scavenger cells (e.g. microglia). Recently, coding sequences (partial cDNA and coding sequence based on a partial gene sequence) of the type II ATPase have been reported. Initial analysis of these sequences revealed an apparent lack of the 5′UTRs. In our attempts to elucidate the promoter, the transcription start site and the 5′ UTR of the APTL II gene, we compared these partial coding sequences to the EST's and genomic databases. This allowed us to elaborate exon/intron organization of ATPase II gene and revealed the possibility of existence of alternative splicing. We then analyzed the region in genomic DNA immediately upstream of the 5′end of the longest 5′EST clone, which revealed the presence of a CpG island. A TATA‐less promoter was predicted. We then amplified the part of the gene containing the promoter by PCR using genomic DNA as template. Cloning and sequencing revealed minor, though significant, differences between our sequence and its counterpart reported by Human Genome Project. Our studies will be followed up by 5′ RACE and analysis of the putative promoter region by its ability to drive an expression of a reporter gene. This will allow us to understand tissue specificity and the functional role of the APTLII enzyme.
N‐acetyl cysteine protects against injury in animal model of transient focal cerebral ischemia
Authors: Sekhon B. K. , Sekhon C. S. , Khan M. , Barbosa E. , Patel S. J. , Singh I. , Singh A. K.
Published: July 2010

Ischemic cerebrovascular disease (stroke) is one of the leading causes of death and long‐term disability worldwide (or developing countries). The purpose of this study is to investigate the therapeutic potential of N‐acetyl cysteine (NAC), a precursor of glutathione, and a potent antioxidant, in tissue protection against transient focal cerebral ischemia model in rats. Our laboratory has already demonstrated the efficacy of NAC against induction of proinflammatory cytokines, iNOS, and production of NO in cultured cells. In this study, 250–300 g male Sprague–Dawley rats were subjected to 45 min of reversible focal cerebral ischemia through middle cerebral artery (MCA) occlusion. The protective effect of NAC is supported by reduced infarct volume as well as the suppression of TNF‐α and iNOS in the treated animals. Pretreatment with NAC reduced the infarct size by 49.6% as compared to the saline treated group along with reducing the neurological evaluation score by more than 50%. Our results suggest that preadministration of NAC attenuates focal cerebral ischemia by protecting cells against free radical damage.

Acknowledgements:  Supported by NIH grants NS‐40144, NS‐40810, NS‐22576, NS‐34741 and NS‐37766.
Up‐regulation of `peripheral‐type' benzodiazepine receptors in the globus pallidus in manganese neurotoxicity
Authors: Hazell A. S. , Normandin L.
Published: July 2010

Increasing evidence suggests that astrocytes play a major role in damage to the nervous system in manganese neurotoxicity (MN). Astrocytes accumulate manganese and sequester the metal in mitochondria where it inhibits oxidative phosphorylation. Chronic exposure to manganese leads to selective neuronal loss in basal ganglia structures such as the globus pallidus together with characteristic astrocytic changes known as Alzheimer type II astrocytosis. In previous reports we have demonstrated that treatment of these cells with manganese leads to increased binding site densities of the ‘peripheral‐type’ benzodiazepine receptor (PTBR) complex (Hazell et al. 1999; Neurosci. Lett. 271, 5–8), found predominantly in astrocytes and an important source of neurosteroid production. In the present study, we have examined the effects of manganese treatment in a subacute rat model of manganese neurotoxicity. Male Sprague–Dawley rats (200 g) were administered manganese (II) chloride (50 mg/kg, i.p. in 0.2 mL saline) daily for 1 and 4 days while control animals were injected with carrier only. Using neutron activation analysis, levels of manganese in the globus pallidus were found to be increased by 81% after 24 h of treatment but were elevated by 551% compared to controls after 4 days. Manganese treatment also led to a 110% increase in levels of the 18 kDa isoquinoline carboxamide‐binding protein, a major constituent of the PTBR, after 4 days but with no change in protein expression at 1 day by immunoblotting, consistent with the neutron activation findings. These results indicate that PTBRs and possibly neurosteroids are an early response to manganese exposure and may play a major role in the pathophysiology of MN.
Zinc causes loss of membrane potential and alters production of reactive oxygen species in isolated brain mitochondria
Authors: Dineley K. E. , Richards L. L. , Votyakova T. V. , Reynolds I. J.
Published: July 2010

Excessive accumulation of intracellular free zinc is lethal to neurons. The precise mechanism by which zinc kills neurons remains unclear, however, a number of reports suggest that zinc may impair neuronal metabolism. The present study examined how zinc affects the activity of isolated rat brain mitochondria. Using spectrofluorometry and polarography, we determined the effects of zinc on mitochondrial membrane potential (Δψ), reactive oxygen species (ROS) generation, and respiration. In mitochondria respiring on either glutamate + malate (G + M), succinate, or glycerol 3‐phosphate (G3P), addition of submicromolar levels of zinc caused dissipation of delta psi. Zinc‐induced depolarization was rapidly reversed by the addition of the membrane‐impermeant metal chelator EGTA. In all three substrate conditions, ruthenium red (200 nm) partially protected mitochondria from zinc‐induced depolarization, suggesting that the calcium uniporter may mediate mitochondrial zinc uptake. Conversely, addition of cyclosporin A (1 µM) afforded no protection, arguing against zinc‐induced permeability transition. ROS production was enhanced by zinc in mitochondria fueled by either G + M or G3P, but zinc inhibited succinate‐driven ROS production. Zinc inhibited respiration in phosphorylating mitochondria regardless of substrate conditions, and zinc also inhibited respiration in uncoupled mitochondria using G + M. Together, these results suggest that zinc has several deleterious effects on mitochondria that could contribute to neuronal injury.

Acknowledgements:  Supported by NS34138 and the American Heart Association.
Glutamate‐mediated CA++ influx and nuclear damage in retinal ganglion cells purified by panning: role of glia
Authors: DeCoster Mark A. , Bazan Nicolas G.
Published: July 2010

The visual acuity decline in glaucoma is due to degeneration of retinal ganglion cells. We used cell panning with the antibody Thy 1.1 to isolate retinal ganglion cells (RGCs) from rat retina. Individual RGCs were extremely sensitive to glutamate, demonstrating sustained calcium influx responses to 100 nm glutamate. We tested unoprostone for its ability to elicit neuroprotection. This docosanoid decreases intraocular pressure in glaucoma patients and protects mixed retinal ganglion cell cultures from glutamate excitotoxicity and calcium influx. In the presence of retinal glia, RGCs responded to 100 and 500 nm glutamate with transient calcium influx, demonstrating glial buffering effects. Pure retinal glial cultures required on average at least 1 mm glutamate to trigger calcium influx, which was transient and oscillating. Unoprostone (10 μm) inhibited calcium influx stimulated by 5 mm glutamate in retinal glial cultures, while DMSO did not. Unoprostone at 1 μm also blocked neuronal calcium responses to 100 nm glutamate in mixed cultures, while DMSO did not. In RGCs isolated by panning, unoprostone blocked calcium responses to 100 nm glutamate in some cells, while DMSO blocked none. Unoprostone also protected RGCs from 20 μm glutamate‐induced toxicity and nuclear damage. We hypothesize that while unoprostone may affect individual RGCs depending on a putative receptor, the presence of retinal glia also plays a key role in neuroprotection and unoprostone action. The use of the cellular model described here and whole retina preparations will be useful to understand the mechanisms of RGC survival.

Acknowledgements:  Support: Novartis Ophthalmics, Basel, Switzerland.
Role of mycotoxin zearalenon poisoning in abnormalities of thromboplastic activity in rats and ways of their correction
Authors: Karageuzyan K. G. , Karagyozyan M. K.
Published: July 2010

Intravenous administration of alcohol solution mycotoxin zearalenon (MZ) in quantity of 5.0–15.0 μg/body weight to 46 white mongrel male rats weighing 180–200 g, significantly shorten the blood coagulation time (BCT) and increase brain and erythrocyte thromboplastic activity (TA). Our previous investigations have shown that neutral phospholipids (NPL) particularly, phosphatidylcholines (PC) and phosphatidylethanolamines (PE) accelerate the blood clotting process (BCP), while acidic phospholipids (APL) mainly phosphatidyserines, play the role of inhibitors of BCP. Brain tissue thromboplastins are the membrane‐bound PL‐dependent enzymes catalysing the process of blood unactive prothrombin transformation into the active thrombin. It is necessary to draw attention to the relationship between the coefficient NPL/APL quota and TA both in normal and pathological conditions. It was demonstrated that modulation of MZ poisoning in rats accompanied by activation of PC biosynthesis. This fact leads us to the conclusion that elevation of PE quantity under the conditions of MZ poisoning is accompanied also with partial involving of them into methylation processes and provides high concentrations of PC. Intramuscular single administration to poisoned rats of 1 mL of 10% water solution of sodium thiosulfate separately and especially in combination with the Ca ions precipitate of dsRNA in concentration 5.0–25.0 mg/kg leads to normalization of all abnormalities mentioned during the period of 2–4 h MZ intoxication.