Melinda West

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis.

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain-of-function mutations in Cu,Zn-superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis-related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A-SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A-SOD1 mice expressed a selective subset of macrophage-typical cytokines (monokines) including interleukin (IL)1alpha, IL1beta and IL1RA at 80 days increasing by 120 days. Contrastingly, T-cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non-transgenic mice but these were not elevated in G93A-SOD1 mice even at 120 days. Apoptosis-related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up-regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)alpha receptor p55 which was up-regulated at 80 days and increased further at 120 days. These data indicate that in the G93A-SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFalpha and its receptors may link inflammation to apoptosis in ALS.

Message and protein-level elevation of tumor necrosis factor α (TNFα) and TNFα-modulating cytokines in spinal cords of the G93A-SOD1 mouse model for amyotrophic lateral sclerosis

Recent data indicate that certain pro-inflammatory cytokines are transcriptionally upregulated in the spinal cords of G93A-SOD1 mice, a model of amyotrophic lateral sclerosis (ALS). We previously showed that the receptor for tumor necrosis factor alpha (TNF-R1) was notably elevated at late presymptomatic as well as symptomatic phases of disease (J. Neurochem. 82 (2002) 365). We now extend these findings by showing that message for TNFalpha, as well as mRNA for interferon gamma (IFNgamma) and transforming growth factor beta1/2 (TGFbeta1, TGFbeta2), is simultaneously increased. Furthermore, TNFalpha protein is significantly increased in G93A-SOD1 mouse spinal cords, as are protein levels for interleukin-6 (IL6), IFNgamma, and the chemokines RANTES (CCL5) and KC. The interaction of TNFalpha, IL6, and IFNgamma proteins was modeled in vitro using Walker EOC-20 murine microglia with nitrite (NO(2)(-)) efflux as a quantitative index of cell response. TNFalpha alone caused robust NO(2)(-) flux, while IL6 had a lesser effect and neither IFNgamma nor IL1beta was active when applied singly. The TNFalpha stimulus was potently magnified in the presence of IL6 or IFNgamma. When applied in combination at very low concentrations, IFNgamma co-synergized with IL6 to produce a multiplicative increase in NO(2)(-) after stimulation with TNFalpha. Taken together, these data suggest that modest increases in multiple synergistic cytokines could produce a disproportionately severe activation of microglia within the degenerating spinal cord. Our data support a model wherein TNFalpha acts as a principal driver for neuroinflammation, while several co-stimulating cytokines and chemokines act to potentiate the TNFalpha effects.

Temporal patterns of cytokine and apoptosis-related gene expression in spinal cords of the G93A-SOD1 mouse model of amyotrophic lateral sclerosis: Gene expression changes in ALS mice

Familial amyotrophic lateral sclerosis (FALS) is often caused by gain‐of‐function mutations in Cu,Zn‐superoxide dismutase (SOD1). Multiprobe ribonuclease protection assays (RPAs) were used to investigate expression of 36 different cytokines and apoptosis‐related genes in spinal cords of mice that ubiquitously express human SOD1 bearing a glycine (r) alanine substitution at residue 93 (G93A‐SOD1). Mice were studied at late presymptomatic stage (80 days), and at 120 days when the animals experience severe hindlimb paralysis and accumulation of oxidatively modified proteins. Spinal cord tissue from G93A‐SOD1 mice expressed a selective subset of macrophage‐typical cytokines (monokines) including interleukin (IL)1α, IL1β and IL1RA at 80 days increasing by 120 days. Contrastingly, T‐cell derived cytokines (lymphokines) including IL2, IL3 and IL4 were detected at low levels in non‐transgenic mice but these were not elevated in G93A‐SOD1 mice even at 120 days. Apoptosis‐related genes were generally unaffected at 80 days but multiple caspases and death receptor components were up‐regulated at 120 days; the only exceptions being FADD and the tumor necrosis factor (TNF)α receptor p55 which was up‐regulated at 80 days and increased further at 120 days. These data indicate that in the G93A‐SOD1 mouse: (i) cytokine expression changes precede bulk protein oxidation and apoptosis gene expression; (ii) lymphocyte contributions to cytokine expression in FALS are likely minor; and (iii) TNFα and its receptors may link inflammation to apoptosis in ALS.

Hydroxyl free radical mediated formation of 8-hydroxyguanine in isolated DNA☆☆☆

Formation of 8-hydroxyguanine within calf thymus DNA has been studied after exposure to uv-H2O2 as a hydroxyl free radical generating system. Using high-pressure liquid chromatography with electrochemical detection, we measured the amount of 8-hydroxy-2-deoxyguanosine (8-OHdG) in the enzymatically digested DNA. The 8-OHdG content of uv-exposed DNA increased linearly with increasing H2O2 levels up to 0.03%, above which the rate of increase was less than linear. All hydroxyl free radical scavengers studied (mannitol, ethanol, thiourea, and salicylate), if present in the system when DNA was exposed to uv-H2O2, caused a decrease in the amount of 8-OHdG formed. Thiourea when incubated with damaged DNA caused a loss of 8-OHdG when it was an integral part of DNA. In contrast, thiourea did not react with the nucleoside free in solution. Reduced glutathione did not cause a decrease of 8-OHdG, either when it was an integral part of DNA, or, as the free nucleoside in solution.

The arachidonic acid 5‐lipoxygenase inhibitor nordihydroguaiaretic acid inhibits tumor necrosis factor α activation of microglia and extends survival of G93A‐SOD1 transgenic mice

Familial forms of amyotrophic lateral sclerosis (ALS) can be caused by mutations in copper, zinc‐superoxide dismutase (SOD1). Mice expressing SOD1 mutants demonstrate a robust neuroinflammatory reaction characterized, in part, by up‐regulation of tumor necrosis factor alpha (TNFα) and its primary receptor TNF‐RI. In an effort to identify small molecule inhibitors of neuroinflammation useful in treatment of ALS, a microglial culture system was established to identify TNFα antagonists. Walker EOC‐20 microglia cells were stimulated with recombinant TNFα, with or without inhibitors, and the cell response was indexed by NO2– output. Three hundred and fifty‐five rationally selected compounds were included in this bioassay. The arachidonic acid 5‐lipoxygenase (5LOX) and tyrosine kinase inhibitor nordihydroguaiaretic acid (NDGA), a natural dicatechol, was one of the most potent non‐cytotoxic antagonists tested (IC50 8 ± 3 μm). Investigation of the G93A‐SOD1 mouse model for ALS revealed increased message and protein levels of 5LOX at 120 days of age. Oral NDGA (2500 p.p.m.) significantly extended lifespan and slowed motor dysfunction in this mouse, when administration was begun relatively late in life (90 days). NDGA extended median total lifespan of G93A‐SOD1 mice by 10%, and life expectancy following start of treatment was extended by 32%. Disease‐associated gliosis and cleaved microtubule‐associated tau protein, an indicator of axon damage, were likewise reduced by NDGA. Thus, TNFα antagonists and especially 5LOX inhibitors might offer new opportunities for treatment of ALS.

Conditions influencing yield and analysis of 8-hydroxy-2′-deoxyguanosine in oxidatively damaged DNA

We have conducted studies to obtain practical knowledge regarding the stability, digestion, and analytical determination of the content of 8-hydroxy-2-deoxy-guanosine (8-OHdG) in oxidatively damaged DNA. Utilizing H2O2 plus uv light to form oxidatively damaged DNA, we found that storage of the DNA at -20 degrees C at alkaline pH caused a significant loss of 8-OHdG, whereas storage at -20 degrees C at neutral or acidic pH prevented loss of 8-OHdG. The 8-OHdG within DNA is stable at 100 degrees C for at least 15 min. Formation of 8-OHdG within DNA using uv light and H2O2 as a hydroxyl free radical-generating system yields the highest amounts when low levels of phosphate buffer are used; but the use of Tris or citrate buffers causes a lower yield of 8-OHdG because these buffers act as scavengers for the hydroxyl free radicals. Independent assessment of hydroxyl free radical flux by the use of salicylate trapping allows assessment of competitive radical reactions. Ethanol washing of plastic microfuge tubes prior to DNA enzymatic digestion improved the yield of 8-OHdG and reduced the variability between samples. Digestion of the oxidatively damaged DNA by the use of a method involving DNase I, endonuclease, phosphodiesterase, and alkaline phosphatase produced the highest yield of 8-OHdG.

Oxidative biochemical markers; clues to understanding aging in long-lived species.

Clues as to why long-lived species live so much longer than short-lived species may reside in the amount of reactive oxygen species (ROS) produced and their effect on damaging cell components (especially proteins) and alterations of crucial cellular processes. Rigorous evaluation of these concepts required critical comparisons of oxidative damage markers and/or parameters with assess difference in ROS flux and the critical age-modifying processes they influence. The limited experimental comparative results available implicate that ROS production per unit weight of total oxygen consumed is much less in the longer-lived species than in shorter-lived species. Mitochondria are the major site of ROS production. They are also the functional nexus for intracellular signaling thus modulating stress and growth factor mediated cellular survival, proliferation and apoptotic processes. Mitochondrial DNA mutations, perhaps caused by ROS, increase with age. Mutant mitochondria possess comparative replicative advantage, which leads to age-specific intracellular swarms. General inflammatory stress tends to increase with age. Disruption in coordinated cell-to-cell signaling triggered by alterations in intracellular signaling may be the basis of the age-related increases in tissue inflammation, which may explain some of the differences between long-lived species and short-lived species.

Region‐selective effects of neuroinflammation and antioxidant treatment on peripheral benzodiazepine receptors and NMDA receptors in the rat brain

Following induction of acute neuroinflammation by intracisternal injection of endotoxin (lipopolysaccharide) in rats, quantitative autoradiography was used to assess the regional level of microglial activation and glutamate (NMDA) receptor binding. The possible protective action of the antioxidant phenyl‐tert‐butyl nitrone in this model was tested by administering the drug in the drinking water for 6 days starting 24 hafter endotoxin injection. Animals were killed 7 days post‐injection and consecutive cryostat brain sections labeled with [3H]PK11195 as a marker of activated microglia and [125I]iodoMK801 as a marker of the open‐channel, activated state of NMDA receptors. Lipopolysaccharide increased [3H]PK11195 binding in the brain, with the largest increases (two‐ to threefold) in temporal and entorhinal cortex, hippocampus, and substantia innominata. A significant (> 50%) decrease in [125I]iodoMK801 binding was found in the same brain regions. Phenyl‐tert‐butyl nitrone treatment resulted in a partial inhibition (approx. 25% decrease) of the lipopolysaccharide‐induced increase in [3H]PK11195 binding but completely reversed the lipopolysaccharide‐induced decrease in [125I]iodoMK80 binding in the entorhinal cortex, hippocampus, and substantia innominata. Loss of NMDA receptor function in cortical and hippocampal regions may contribute to the cognitive deficits observed in diseases with a neuroinflammatory component, such as meningitis or Alzheimers disease.

Mediation of 8-hydroxy-guanine formation in DNA by thiazin dyes plus light

We have discovered that methylene blue plus light mediates the formation of 8-OHdG in DNA. Methylene blue is one of several thiazin dyes and we report here that the other thiazin dyes tested, in combination with white light, are effective in mediating 8-OHdG formation in DNA. The effectiveness of light plus the thiazin dyes in forming 8-OHdG in DNA were as follows: methylene blue greater than azure B greater than azure A greater than toluidine blue greater than thionin. Two other compounds tested; riboflavin and fuschin acid, in combination with light, caused formation of very little, if any, 8-OHdG in DNA. Thiazin dye mediated formation of 8-OHdG in DNA was not inhibited by the spin trap alpha-phenyl-t-butyl nitrone, which supports our previous observations that oxygen free radical scavengers did not inhibit methylene blue plus light mediated 8-OHdG formation in DNA. Ascorbate addition to methylene blue plus DNA, in the absence of light, was ineffective in mediating 8-OHdG formation in DNA.

Myelin Proteolipid Protein: Function in Myelin Structure Is Distinct from Its Role in Oligodendrocyte Development

The myelin proteolipid proteins PLP and DM20 are essential for the compaction of central nervous system myelin and they play an important role in the maturation of the oligodendrocyte. The specific function of the less abundant DM20 isoform is still unknown, but rescue experiments previously indicated that both isoforms are necessary for oligodendrocyte maturation. In vitro experiments have suggested DM20 may assist in the translocation of PLP into the membrane. We tested this hypothesis in vivo, by investigating whether wild-type PLP derived from a transgene could be incorporated into the myelin membrane of Plp mutant rumpshaker mice. We previously demonstrated that expression of the PLP transgene alone in a more severe Plp mutant, jimpy mouse, did not result in PLP incorporation into the myelin. Here we report that there was significantly more PLP in white matter from rumpshaker expressing the PLP transgene than their nontransgenic rumpshaker littermates and that myelin structure was improved. The delay in oligodendrocyte development was not alleviated by expression of the PLP transgene however, supporting an essential role for DM20 in oligodendrocyte maturation.