Emerging evidence suggests the involvement of programmed cell death and inflammation in amyotrophic lateral sclerosis (ALS). To assess molecular pathological effects of the anti-inflammatory peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist pioglitazone in ALS, we verified changes in the population of neurons, astrocytes, and microglia in the ventral horns of spinal cord lumbar segments from the pioglitazone-treated and non-treated groups of mice carrying a transgene for G93A mutant human superoxide dismutase-1 (SOD1) (ALS mice) and non-transgenic littermates (control mice), performed immunohistochemical and immunoblot analyses of PPARgamma, active form of phosphorylated p38 mitogen-activated protein kinase (p-p38) and inhibitor of nuclear factor-kappaB (NF-kappaB)-alpha (IkappaBalpha) in the spinal cords, and compared the results between the different groups. Image analysis revealed that optical density of NeuN-immunoreactive neurons was significantly lower in the non-treated groups of presymptomatic and advanced ALS mice than in the non-treated groups of age-matched control mice and was recovered with pioglitazone treatment, and that optical densities of GFAP-immunoreactive astrocytes and Iba1-immunoreactive microglia were significantly higher in the non-treated group of advanced ALS mice than in the non-treated group of control mice and were recovered with pioglitazone treatment. Immunohistochemical analysis demonstrated that immunoreactivities for PPARgamma and p-p38 were mainly localized in neurons, and that IkappaBalpha immunoreactivity was mainly localized in astrocytes and microglia. Immunoblot analysis showed that pioglitazone treatment resulted in no significant change in nuclear PPARgamma-immunoreactive density, a significant decrease in cytosolic p-p38-immunoreactive density, and a significant increase in cytosolic IkappaBalpha-immunoreactive density. Our results suggest that pioglitazone protects motor neurons against p38-mediated neuronal death and NF-kappaB-mediated glial inflammation via a PPARgamma-independent mechanism.

BackgroundEmerging evidence suggests that innate immunity and increased oxidative stress contribute to pathomechanisms in amyotrophic lateral sclerosis (ALS). The aim of the present study was to verify the involvement of monocyte chemoattractant protein-1 (MCP-1) and its specific CC chemokine receptor 2 (CCR2) in the disease progression of ALS. We here demonstrate the expression state of MCP-1 and CCR2 in lumbar spinal cords of mice overexpressing a transgene for G93A mutant human superoxide dismutase 1 (SOD1) (ALS mice) as a mouse model of ALS as well as the involvement of MCP-1/CCR2-mediated signaling in behavior of cultured astrocytes derived from those mice.ResultsQuantitative polymerase chain reaction analysis revealed that MCP-1 and CCR2 mRNA levels were significantly higher in ALS mice than those in nontransgenic littermates (control mice) at the presymptomatic stage. Immunoblot analysis disclosed a significantly higher CCR2/β-actin optical density ratio in the postsymptomatic ALS mouse group than those in the age-matched control mouse group. Immunohistochemically, MCP-1 determinants were mainly localized in motor neurons, while CCR2 determinants were exclusively localized in reactive astrocytes. Primary cultures of astrocytes derived from ALS mice showed a significant increase in proliferation activity under recombinant murine MCP-1 stimuli as compared to those from control mice.ConclusionsOur results provide in vivo and in vitro evidence that MCP-1 stimulates astrocytes via CCR2 to induce astrocytosis in ALS with SOD1 gene mutation. Thus, it is likely that MCP-1/CCR2-mediated sigaling is involved in the disease progression of ALS.

Several studies have documented the involvement of oxidative stress represented by lipid peroxidation in the pathogenesis of Alzheimer's disease (AD). To test whether the highly reactive carbonyl crotonaldehyde (CRA), generated during lipid peroxidation, is involved in AD, we performed an immunohistochemical analysis in AD and age-matched control hippocampi using a specific antibody against protein-bound CRA (P-CRA). In the AD cases, P-CRA immunoreactivity was preferentially localized in reactive astrocytes and microglia around senile plaques (SPs) and those present in the neuropil, while it was weakly detectable in neurons and neurofibrillary tangles. P-CRA immunoreactivity was also localized in all portions of diffuse SPs and the dystrophic neurites of neuritic and classical SPs, but was undetectable in amyloid cores. Age-matched controls showed P-CRA immunoreactivity only very weakly in neurons. In contrast to P-CRA, immunoreactivities for protein-bound acrolein and 4-hydroxy-2-nonenal were mainly localized to neurons and rarely seen in glial cells. Our results suggest that increased oxidative stress and CRA formation in glial cells is implicated in the disease processes of AD.

These results suggest that Smad4 is essential for a subset of, but not all, TGF-β/BMP-dependent developmental processes in the embryonic retina. In addition, genetic requirements for Smad4 in the embryonic retina are evident predominantly in the developmental events regulated by the BMP branch of the TGF-β signaling pathway.

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