Fig 2. Axotomy-induced mitochondrial fission inhibits neuroprotection.

(A and A’) Representative images of dendritic mitochondria in control (A) and Drp1 RNAi (A’) neurons before and 8h post axon injury (hpa) are shown. mito-GFP and mCD8-RFP were coexpressed in ddaE neurons under the control of 221-Gal4 in order to visualize mitochondria and the cell membrane, respectively. Orange, blue and magenta arrows indicate long (>1.5 μm), medium (1–1.5 μm) and short (<1 μm) mitochondria respectively. Scale bars are 10 μm. (B-D’) The length, length distribution, and total number of mitochondria in control (B-D) and Drp1 RNAi neurons (B’-D’) before and after axon injury were measured. Statistical significance was determined using a Fisher’s exact test (C and C’), or a t test (B, B’, D and D’). * p<0.05, ** p<0.01, *** p<0.001, N.S. not significant. Error bars represent SD. (B, B’, D and D’) The numbers of neurons analyzed are indicated above the bars. (C) 227 and 229 mitochondria from 9 neurons were analyzed for uninjured and 8hpa, respectively. (C’) 173 and 169 mitochondria from 8 neurons were analyzed for uninjured and 8hpa, respectively. (E) The NP assay was performed in Drp1 RNAi neurons labeled with EB1-GFP. Red arrows indicate the site of axon injury and purple arrows, dendrite injury. Green lines mark stabilized dendrites. Scale bar, 20 μm. (F) Quantification of NP is shown with control data from Fig 1D for comparison. * p<0.05 and **p<0.01, determined by Fisher’s exact test. The numbers of neurons are indicated above the bars. For the RNAi experiment, Rtnl2 data is shown as the matched control and yw (no RNAi hairpin) control data was used for the overexpression comparison. (G) Dendrite injury was performed in Drp1 RNAi neurons without pre-axotomy. Dendrite degeneration assayed at the indicated times. Control data from Fig 1E is included for comparison. The numbers of cells analyzed for each condition are shown above the bars.