Samples were analyzed by Western blotting. Culture and Analysis of MEFs. GM130 have yet to be explored in a mammal. Here, we generated GM130 knockout (KO) mice and investigated the consequences of GM130 loss upon Golgi architecture and function within the nervous system. We find that loss of GM130 leads to disrupted business and altered positioning of the Golgi apparatus in cerebellar Purkinje cells, which is usually accompanied by impaired polarized trafficking to the apical dendrite. Importantly, we find that these cellular defects manifest as a loss of Purkinje cell viability and progressive cerebellar atrophy, leading to ataxia. Our findings therefore indicate that disruption of the Golgi apparatus and impairment of secretory trafficking result in neuronal loss in vivo and thus may contribute to the phenotypes observed in neurodevelopmental and neurodegenerative disease. Results Generation of GM130 KO Mice. To determine the physiological importance of GM130 in vivo, we generated a global KO mouse (mice, which lacked detectable GM130 (Fig. 1and = 20), = 41), and = 21) mice. ** 0.01. ( 0.01. Open in a separate windows Fig. S1. Generation of KO mice. (KO mice. The genomic structure of the mouse gene (first line), illustrations of the targeting vector (second line), the resultant targeted allele (third line), and the genomic deleted allele (fourth line) are shown. (with mice bearing a transgene, which is usually expressed throughout the nervous system (29), the neuron-specific KO offspring ([control mice (Ctrl)] littermates up to 1 1.5 y RN of age. The growth retardation observed in is usually active. Open in a separate windows Fig. S2. Western NKY 80 blotting for GM130 in tissue-specific KO mice. Protein lysates from different organs of control (Ctrl) and tissue-specific KO mice were immunoblotted with anti-GM130 and anti-GAPDH antibodies. GM130 is not expressed in the lungs of mice displayed a striking ataxia phenotype (Movie S1 and Fig. S3mice, and transgenic mice and mice did not display any motor abnormalities. To assess motor coordination quantitatively, the and Fig. S3 and = 5, ** 0.01). (and = 7 control mice, = 8 0.05, ** 0.01. Results from four impartial trials are shown. Data are presented as the mean SEM. (and = 9 control mice, = 9 0.05, ** 0.01. Results from three impartial trials are shown. Data are presented as the mean SEM. Open in a separate windows Fig. S3. Motor deficits of GM130 KO mice. Motor coordination performance on a rotarod with slow acceleration from 4 to 40 rpm over 5 min was assessed in WT control and = 4; = 4) (control and and = 7; = 8) and for = 9; = 9). * 0.05, ** 0.01. Data are presented as the mean SEM. Progressive NKY 80 Cerebellar Atrophy and Purkinje Cell Loss in and and and and indicate the position of the cerebellum. (Scale bar in indicate Purkinje cells. The granule cell layer (GL) and molecular layer (ML) are indicated. (Scale bar in and = 3; ** 0.01. Data are presented as the mean SD. (and = 3; * 0.05, ** 0.01. Data are presented as the mean SD. (= 3; * 0.05. Data are presented as the mean SD. Open in a separate windows Fig. S5. Purkinje cell NKY 80 apoptosis in the cerebellum of GM130 KO mice. (20 m and 10 m.) (= 3; *** 0.001. Data are presented as mean SD. (side of the image. (Scale bar, 500 m and 50 m.) Disruption of Golgi Architecture and Positioning upon GM130 KO. Studies in cultured cells have revealed a role for GM130 in maintaining mammalian Golgi ribbon organization and pericentrosomal positioning (25, 31). GM130 also participates in vesicle tethering during ER-to-Golgi traffic (24, 26, 27), and can function as a scaffold for activation of Cdc42 or Stk25 that is relevant for cell migration (32C34). To elucidate the cellular basis of the ataxic phenotype and Purkinje cell degeneration of and and and see Fig. S8). Changes in Golgi ultrastructure in the Purkinje cells were clearly observed using transmission electron microscopy with.