We found that pretreatment of cultured neurons with the CaM kinase II/IV inhibitor KN-93 blocks Golgi fragmentation by bicuculline treatment

We found that pretreatment of cultured neurons with the CaM kinase II/IV inhibitor KN-93 blocks Golgi fragmentation by bicuculline treatment. 10 DIV, = 8; 14 DIV, = 9; 17 DIV, = 17). Golgi Fragmentation also Results from Neuronal Hyperactivity. Knowing that Golgi fragmentation results from neuronal hyperexcitability, we wondered if hyperactivity also causes fragmentation of the Golgi complex. Mature cultured neurons (21 DIV) were treated with bicuculline for 1C2 d, then bicuculline was removed (Fig. 2= 10; Bic, 1 d, = 7; Bic, 3 d, = 6; APV, 1 d, ICA-110381 = 9; APV, 3d, = 7; TTX+Bic, 1 d, = 5). For Bic, 1 d, < 0.1. A similar observation ICA-110381 of Golgi fragmentation was detected for neurons after removal of APV (Fig. 2region of the Golgi, TGN38 is located in the Golgi. For TGN38, the number of Golgi fragments was 14 (IR = 11C17) after 1 d bicuculline treatment, 16 (IR = 11C22) for 1 d after APV withdrawal Mouse monoclonal to Plasma kallikrein3 (APV, 3 d), and 4 (IR = 2C5) for untreated control (Fig. 3= 10; Bic, 1 d, = 9; APV, 1 d, = 8; APV, 3d, = 7). Golgi Fragmentation from Hyperactivity Is Reversible. The experiments shown in Figs. 2 and ?and33 suggest that the Golgi fragmentation is reversible upon return to normal neuronal activity. Additionally, we checked the neurons during Golgi fragmentation conditions (both during bicuculline and after APV washout) for signs of apoptosis and found the neurons remain healthy with intact mitochondria and nuclei (Fig. S2). Nonetheless, we wanted to observe the reversibility of the Golgi fragmentation, so we turned to live cell time-lapse imaging of cultured hippocampal neurons cotransfected with fluorescently labeled Golgi enzyme Mgat2 (Mgat2CEGFP) and myristoylated Td-Tomato (to visualize neuron morphology). The somatic region of individual neurons was imaged before and 1 d after treatment with bicuculline. With addition of bicuculline, Mgat2CEGFP localization showed some fragmentation (Fig. 4shows two example neurons and Fig. 4shows a mock-treated control). After 1 d of bicuculline treatment, the medium was removed and replaced with preconditioned normal medium. Following return to normal medium, the neurons were imaged 2 d later to observe reversal of the Golgi fragmentation. The summary data of individual neurons (Fig. 4= 12) and control (black, = 4) neurons. Activity-Dependent Golgi Fragmentation Requires CaM Kinase Activation. Knowing increased neuronal activity leads to an increase in intracellular calcium, we hypothesized that a calcium-dependent pathway may lead to the Golgi fragmentation. We found that pretreatment of cultured neurons with the CaM kinase II/IV inhibitor KN-93 blocks Golgi fragmentation by bicuculline treatment. Using the same conditions of mature cultured hippocampal neurons as used in Fig. 1, KN-93 was added 20 min before addition of bicuculline (Fig. 5and = 10; Bic, 1 d, = 7; KN-93+Bic, 1 d, = 5; Bic, 3 d, = 6). (and = 11; OA, 1 d, = 10; OA, 2 d, = 9). (and Fig. S1) are color-coded (only for ease of visualization of fragments) with spectrum coloring of red (largest fragment) to violet (for the smallest). Live Time-Lapse Imaging. Cotransfected neurons (Mgat2-EGFP and myristoylated Td-Tomato) were imaged by ICA-110381 epifluorescence microscopy at 15 DIV. Then at least half of the conditioned medium was removed and saved, and bicuculline (20 M) was added to the cells. After 1 d, the same neurons were imaged before removal of the bicuculline-containing medium and replacement with the conditioned medium. Two days later the cells were imaged again. The numbers of distinct fragments of Mgat2CEGFP signal were counted and compared with mock-treated cultures. Data Analysis. Results are reported as median and IR; means and SD were not used, as the datasets are not normally distributed. Comparisons of group medians were performed with nonparametric KruskalCWallis with Dunns posttest using Prism 5 (GraphPad Software), with differences considered significant at < 0.05 (*< 0.05, **< 0.01, ***< 0.001 in all graphs). Supplementary Material Supporting Information: Click here to view. Acknowledgments This work was supported by a National Institutes of Health National Research Service Award postdoctoral fellowship (to D.A.T.) and National Institute of Mental Health Grant MH065334. Y.N.J. and L.Y.J. are Howard Hughes ICA-110381 Medical Institute investigators. Footnotes The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1220978110/-/DCSupplemental..