Figure 2.

MPP⁺ rapidly decreases mitochondrial movement in DA axons as shown by mtDendra2. A, Diagram of microchamber device. B, Transmitted light image of segregated axons derived from DIV12 DA/GFP cultures in the top panel, GFP fluorescence in the middle panel, and the merged image in the bottom panel showing TH-positive and TH-negative axons in same field. C, Immunostaining of the axonal side with TH, the axonal marker Tau, and the dendritic marker MAP2. Tau but not MAP2-positive processes is present on axonal side. Scale bar, 10 μm. D, mtDendra2 colocalizes with TMRE. Despite presence of nonconverted mtDendra2, DA axon is easily identified. E, Axonal movement of mitochondria. Mitochondria labeled with mtDendra2 were imaged for 5 min at 5 s intervals after 30 min incubation with and without 2 μm MPP⁺. For consistency, mitochondrial measurements were assessed near the axon terminal at least 2 mm away from the cell bodies. Resulting kymographs are shown below. F, Number of moving mitochondria per 100 μm length of axon was calculated. G, Speed was calculated as described in Materials and Methods. F, G, Mean ± SEM, *p < 0.05, total of 17 (control) and 14 (MPP⁺-treated) axons derived from at least 7 dishes in 3 independent experiments. The anterograde speed (Antero) was decreased and the retrograde speed (Retro) increased as early as 30 min after MPP⁺ treatment. Scale bars: B–D, 10 μm. Hatching indicates toxin treatment.