Shenyun Mou

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.

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.