[Show abstract][Hide abstract] ABSTRACT: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which glial cells play significant roles in disease progression. Connexins (Cxs) form homotypic or heterotypic gap junctions (GJs) and allow direct intercellular communications among nervous tissue cells. The role of Cxs in motor neuron disease has never been investigated; therefore, we aimed to evaluate alterations of Cxs in mSOD1-transgenic (mSOD1-Tg) mice in comparison with their non-transgenic (non-Tg) littermates at the same ages.
We pathologically evaluated temporal changes to astrocytic Cx43/Cx30 and oligodendrocytic Cx47/Cx32 immunoreactivities at presymptomatic, disease-progressive, and end stages, relative to aquaporin-4 (AQP4), glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-2 (EAAT2), myelin-oligodendrocyte glycoprotein (MOG), and Nogo-A immunoreactivities, and observed neuronal loss by NeuN and neurofilament immunostaining, and microglial response by Iba-1 immunostaining. We also performed quantitative immunoblotting and real-time PCR analyses for Cxs.
The mSOD1-Tg mice showed neuronal and axonal loss in the anterior horns of the lumbar spinal cord accompanied by increased activation of microglia compared with non-Tg mice at the disease-progressive and end stages. Expression patterns of Cxs were not different between mSOD1-Tg and non-Tg mice at the presymptomatic stage, but immunoreactivities for GFAP, Cx43, Cx30 and AQP4 were increased in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. By contrast, Cx47 and Cx32 immunoreactivities were markedly diminished in Nogo-A-positive oligodendrocytes in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages, especially in oligodendrocytes showing SOD1 accumulation. EAAT2 immunoreactivity was also diminished in the anterior horns of mSOD1-Tg mice at the disease-progressive and end stages. Quantitative immunoblotting revealed a significant reduction in Cx47 and Cx32 protein levels in mSOD1-Tg mice at the disease-progressive and end stages. The levels of Cx47 and Cx32 mRNAs were also decreased at these stages.
Our findings indicate that oligodendrocytic and astrocytic GJ proteins in the anterior horns of spinal cord in mSOD1-Tg mice are profoundly affected at the disease-progressive and end stages, where disruption of GJs among glial cells may exacerbate motor neuronal death.
Journal of Neuroinflammation 03/2014; 11(1):42. DOI:10.1186/1742-2094-11-42 · 5.41 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanisms underlying abnormal vascular endothelial growth factor (VEGF) production in amyotrophic lateral sclerosis (ALS). We immunohistochemically studied VEGF, its receptors VEGFR1 and 2, and hypoxia-inducible factor-1α (HIF-1α) in autopsied ALS spinal cords. We also chronologically assessed expression of HIF-1α, karyopherin β1, karyopherin β-cargo protein complex inhibitors, and nuclear pore complex proteins in G93A mutant superoxide dismutase 1 (mSOD1) transgenic mice at presymptomatic, symptomatic and end stages. In ALS patients, compared with controls, HIF-1α immunoreactivity in the cytoplasm of anterior horn cells (AHCs) was significantly increased, while immunoreactivities for VEGF and VEGFRs were significantly decreased. Similar changes in HIF-1α and VEGF levels were observed in mSOD1 transgenic mice. HIF-1α colocalized with karyopherin β1 in the cytoplasm of AHCs and karyopherin β1 colocalized with nucleoporin 62 (Nup62) on the nuclear envelope. From the presymptomatic stage of mSOD1 transgenic mice, karyopherin β1 immunoreactivity in AHC nuclei significantly deceased and morphological irregularities of the Nup62-immunostained nuclear envelope became more pronounced with disease progression. Thus, in AHCs from mSOD1 transgenic mice, transport of cytoplasmic HIF-1α to the nuclear envelope and into the nucleus is impaired from the presymptomatic stage, suggesting that impaired cytoplasmic-nuclear transport of HIF-1α through the nuclear pore might precede motor neuron degeneration.
[Show abstract][Hide abstract] ABSTRACT: Non-cell-autonomous motor neuronal death is suggested in a mutant Cu/Zn superoxide dismutase 1 (mSOD1)-mediated amyotrophic lateral sclerosis (ALS) model, in which microglia and T cells play significant roles in disease progression. However, it remains unknown whether these cells are toxic or protective. The present study aimed to clarify the developmental age-related alterations of neuronal, glial and T cell responses to acute neuron injury in non-transgenic (N-Tg) mice, and the in vivo effects of mSOD1 on these changes by studying N-Tg and mSOD1-Tg mice subjected to unilateral hypoglossal nerve axotomy at young (8 weeks) and adult (17 weeks) ages. Adult N-Tg mice showed increased neuronal viability on day 21 after axotomy and trends toward increased numbers of recruited microglia on day 3 and T cells on day 7, in the hypoglossal nucleus, compared with young N-Tg mice. Quantitative comparisons between mSOD1-Tg and N-Tg mice at the same ages, on day 3 after axotomy, showed that microglial recruitment was significantly lower in mSOD1-Tg mice than in 17-week-old N-Tg mice (the disease progression stage), but the same difference was not seen in 8-week-old mice (the presymptomatic stage), despite good preservation of hypoglossal neurons. Infiltration of CD3-positive T cells, mostly CD4-positive, on day 7 and the viability rate of hypoglossal neurons on the operated side compared with the contralateral side on day 21 were significantly decreased in mSOD1-Tg mice compared with N-Tg mice aged 17 weeks, but the same difference was not seen in mice aged 8 weeks. On day 3 after axotomy, expression levels of IGF-1 mRNA in the operated hypoglossal nucleus were significantly lower in mSOD1-Tg mice than N-Tg mice at 17 weeks of age. The observation that depressed microglial and T cell responses and expression of neurotrophic factors coincided with reduced neuronal viability in adult mSOD1-Tg mice suggests that diminished neuroprotective functions of mSOD1 microglia and T cells may contribute to exaggerated neuronal death.