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. 2014 Mar 19;34(12):4167–4174. doi: 10.1523/JNEUROSCI.2350-13.2014

Figure 2.

Figure 2.

Effects of ALS-linked mutations on the characteristics of TDP-43-enriched RNA granules. A, EGFP-TDP-43 (WT, A315T, Q343R, or K82/84A) was coexpressed with mCherry in DIV 10 rat hippocampal primary neurons. TDP-43 formed RNA granules in neuronal processes. mCherry fluorescence was expressed in the whole neuron and thus was used as a marker to highlight neuronal processes for the quantification of TDP-43 RNA granule distribution density and the furthest neuronal process distance of TDP-43 RNA granules. Scale bar, 10 μm. B, Representative hippocampal neuronal processes expressing TDP-43-enriched RNA granules: WT TDP-43, A315T, and Q343R (ALS-linked TDP-43 mutations) and K82/84A TDP-43 (a mutation removing the nuclear localization signal). Scale bar, 5 μm. C, Quantification of the average size of TDP-43 RNA granules. RNA granules containing TDP-43 with ALS-linked mutations are larger than those containing WT or K82/84A TDP-43. The average granule size was calculated from >500 RNA granules collected from 10 neurons of three independent experiments. *p = 0.01 WT TDP-43 vs K82/84A TDP-43; **p = 0.001 A315T or Q343R vs WT TDP-43. D, Quantification of the average distribution density of TDP-43-enriched RNA granules. The average granule density was calculated from >30 processes of 12 different primary hippocampal neurons. ALS-linked mutations significantly reduced the distribution density of RNA granules in neuronal processes. *p = 0.01 WT vs K82/84A TDP-43; **p = 0.001 A315T or Q343R vs WT TDP-43. Statistical analysis was by one-way ANOVA with Dunnett's posttest. Error bars indicate SEM. E, Quantification of the relative fluorescence intensities of TDP-43 RNA granules in neuronal processes. Data were collected from >600 RNA granules from three independent experiments. F, FRAP analysis of TDP-43 molecules in RNA granules. Nine primary hippocampal neurons were investigated. The average fluorescence intensities of the irradiated TDP-43 RNA granules after photobleaching of from a representative neuron were plotted over recovery time. The curves were fitted by one-phase exponential equations. The calculated mobile fraction of mutant TDP-enriched RNA granules is lower than those RNA granules containing WT TDP-43.