Astrocytic expression of human α-synuclein in mice. (A). Western blot analysis of endogenous α-syn expression in primary cultured cortical neurons and astrocytes. Protein extracts (8 μg) from cultured cortical neurons and astrocytes were immunoblotted using antibodies against human/mouse α-syn (C20), β III-tubulin (neuronal marker), and GFAP (astrocyte maker). β-actin serves as the loading control. (B) A schematic outline of the "tet-off" system for astrocytic expression of α-syn in mice. The tetracycline operator-controlled human α-syn responder mice (tetO-α-syn) were crossed with GFAP-tTA activator mice to generate GFAP-tTA/tetO-α-syn double transgenic (A53T) mice, which selectively expressed exogenous human α-syn in astrocytes. (C) Western blot analysis of human α-syn expression in transgenic mice. One wild-type (WT) and two A53T lines (A8 and E2) of transgenic mice were obtained. The expression of exogenous human α-syn was detected by a human α-syn-specific antibody, α-syn (211). (D) The expression pattern of human α-syn in the brain of A53T (E2 line) transgenic mice revealed by Western blot using both human-specific and human/mouse α-syn antibodies. BS: brainstem, OB: olfactory bulb, Ctx: cortex, ST: striatum, Hip: hippocampus, MB: midbrain, CB: cerebellum; SC: spinal cord. (E-H) Representative images show human α-syn (red) and GFAP (green) co-staining in the striatum of A53T mice. The α-syn immunoreactivity is restricted to GFAP-expressing astrocytes. Scale bar: 20 μm.