Figure 11.

SLC30A10 protects primary midbrain neurons from Mn-induced neurodegeneration. A, Primary midbrain neurons were generated as described in Materials and Methods. Four days after plating, cultures were transfected with FLAG-tagged SLC30A10–WT or Δ105-107. Two days after transfection, cultures were fixed and stained to detect FLAG using a monoclonal antibody and calnexin using a polyclonal antibody. FLAG expression in the cell body appeared to be stronger than in neurites. Scale bar, 10 μm. B, Primary midbrain neurons were generated and transfected with FLAG-tagged SLC30A10 constructs as described above. One day after transfection, cultures were treated with 0 (Control) or 200 μm Mn for 16 h. Cultures were then fixed and stained using a monoclonal antibody against tubulin to visualize neurites and a polyclonal antibody against FLAG to detect transfection. Expression of the FLAG-tagged SLC30A10 constructs in the cells depicted in this panel is shown in Figure 12. For the bottom row, the tubulin images were converted to grayscale, and primary (red), secondary (blue), and tertiary (yellow) neurites that emanated from the neurons were drawn using the NeuronJ plugin of NIH ImageJ. Scale bar, 10 μm. C–F, Quantification of the morphology of neurites from B above. For each neuron, the length of the longest primary neurite, total length of the neuritic tree, total number of primary neurites, and total number of neurites were calculated using the NeuronJ plugin of NIH ImageJ (for each parameter measured, n = 15–25 neurons for each transfection and Mn treatment condition; *p < 0.05 for the difference between Mn-treated cells expressing SLC30A10–Δ105-107 and the other transfection and Mn treatment conditions using one-way ANOVA, followed by Tukey–Kramer post hoc test). G, Primary midbrain neurons were generated as described above and stained to detect tubulin using a monoclonal antibody and GAD 65/67 using a polyclonal antibody. Scale bar, 10 μm.