Elevation of GM2 ganglioside during ethanol‐induced apoptotic neurodegeneration in the developing mouse brain

Abstract

GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase-3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase-3 activation in the 7-day–old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration.

Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

“…2A ), 90 ± 3.3% (mean ± SD from three animals) of the punctate GM2 immunostaining in Area II was found in astrocytes at 24 h. This GM2 staining in Area II also colocalized with CatD. In the control brain, GM2 staining was hardly detected, as previously reported ( 16 ), and the number of GFAP + astrocytes appeared less compared with that of ethanol-treated brain in both Areas I and II ( Fig. 2D ).…”

“…The punctate GM2 immunostaining found 32 h after ethanol exposure was frequently colocalized with LAMP1 and CatD immunolabeling ( Fig. 2C ), suggesting that GM2 accumulated in lysosomes/late phagosomes/late endosomes in astrocytes, as previously shown for activated microglia ( 16 ), because 93 ± 1.7% (mean ± SD of four animals) of GM2 + punctate immunostaining was localized in GFAP + cells at this time point. The ethanol-induced staining for GM2 in astrocytes was observed not only in regions enriched in activated microglia/degenerating neurons, such as Area I, but also in the deep cortical layer/white matter near the cingulum and external capsule (Area II in Fig.…”

“…We found that ethanol increased the number of glial fi brillary acidic protein-positive (GFAP + ) astrocytes in WT mice, in which punctate immunostaining of GM2 was detected in addition to previously reported GM2 accumulation in neurons and activated microglia ( 16 ). In the GalNAcT KO mice, while ethanol still induced neurodegeneration and accumulation of GD3 and GM3 in activated microglia, the increase in GFAP + astrocytes was now abolished.…”

“…We have reported previously that ethanol exposure in P7 mice, which induces widespread apoptotic neurodegeneration and microglial activation within a day (12)(13)(14), increases GM2 ganglioside mainly in lysosomes/late endosomes of activated microglia ( 16 ), which appear to engulf degenerating neurons ( 14 ). Neurodegeneration was detected by FJ staining in many brain areas, including layers IV/V of the primary somatosensory cortex and the cingulate/ retrosplenial cortex 24 h after ethanol exposure in P7 mice ( Fig.…”

“…The fi xed brains or hemispheres were transferred to phosphate-buffered saline solution and kept at 4°C for 2-5 days until cutting 50 m-thick vibratome sections. The free-fl oating sections were immunostained using anti-GM2 antibody (a mouse detected in mitochondria and lysosomes in neurons, and GM2, as well as GD3 ganglioside, enhances cytochrome c release from isolated mitochondria ( 16 ), suggesting that GM2 may be involved in mitochondria-mediated apoptosis, as indicated for GD3 in CD95/Fas-mediated apoptosis in lymphocytes ( 21 ).…”