Fig 2: Comparing the effect of Lis1 KO in brainstem and heart. A, Sagittal brain sections of Lis1 KO mice 5 d after the initial injection of either five injections of 2 mg tamoxifen (top) or two injections of 8 mg tamoxifen (bottom). The 2 × 8 mg treatment resulted in much higher GFP expression than the 5 × 2 mg treatment, particularly in the brainstem and cerebellum. B, Lis1 mRNA levels normalized to B2M mRNA levels from brainstem of no Cre control mice injected with 2 × 8 mg tamoxifen (CON), and Lis1 KO mice injected with either 5 × 2 or 2 × 8 mg tamoxifen. Lis1 mRNA levels were significantly decreased in brainstem of 2 × 8 mg animals, but not 5 × 2 mg animals, relative to no Cre controls, 5 d after initial injection. C, Sections of heart from 5 × 2 mg (top)- and 2 × 8 mg (bottom)-treated Lis1 KO mice. Both the 2 × 8 and 5 × 2 mg treatments resulted in similar levels of GFP expression in heart. D, Lis1 mRNA levels normalized to B2M mRNA levels from heart of 2 × 8 mg-injected no Cre control (CON)-, 5 × 2 mg-, and 2 × 8 mg-treated mice. Lis1 mRNA levels were reduced significantly in both the 5 × 2 mg- and 2 × 8 mg-treated mice relative to the no Cre control but were not significantly different from each other. E, Western blotting of brainstem and heart lysates from cardiomyocyte-specific Myh6 KO mice show reduced levels of Lis1 protein in heart, but not brainstem compared to no Cre control mice (CON). Dynein intermediate chain (DIC) was used as a loading control. F, Whole mount brain (right) and heart (left) from Myh6 KO mouse show recombination (GFP) in heart but not brain. Data in A, C, E, F are representative images from N = 3 mice for each genotype. The RNA quantification in B, D represent mean of data from N = 3 animals of each treatment and genotype ± SD. Significance in B, D determined by one-way ANOVA; *p < 0.05, ***p < 0.001 (see Table 2 for details). Scale bars: 5 mm (A, C) and 2 mm (F).

Fig 3: More mitochondria are observed in sciatic nerve axonal cross-section from LIS1+/– mice or rat nerves exposed to Ciliobrevin-D. (A) Representative electron micrographs of cross-sections of myelinated axons in sciatic nerves from adult WT LIS1 and LIS1+/– mice at mid-thigh. Red arrows point to mitochondria within the axoplasm. EM images from three different mice of each genotype were analyzed for numbers of mitochondria per axon. (B) The pie graphs show the percentage of all axonal cross-sections that contained the indicated number of mitochondria (1- >6). A total of 676 LIS1+/+ axons and 405+/– axons from three mice of each genotype were included, approximately equal numbers per animal. The variation is not because of degeneration but simply a matter of number of axons in each EM micrograph, which was variable. (C) The bar graph shows the mean of data (+/– 95% CI) from the three mice in each category. Axons with 0 mitochondria were excluded because the numbers were large and the differences in one mitochondrion, two mitochondria, or >2 mitochondria were less clear. Significance determined by ANOVA using Holm-Šídák’s multiple comparisons test. ****p < 0.0001; *p > 0.05. (D) Ciliobrevin-D (CB-D) was used to inhibit dynein by placing PEVA nerve cuffs impregnated with the compound around adult rat sciatic nerve in mid-thigh. After 48 h dynein had accumulated under a CB-D cuff but not a DMSO control cuff as seen in the IP-Western for dynein intermediate chain (DIC). (E) Brightfield image of nerve sections stained for EM shows that the nerves look normal and have similar numbers of axons. (F) Unmyelinated axons in Remak bundles also appeared healthy in both DMSO and CB-D treated nerves. (G) The pie graphs show the percentage of all axonal cross-sections that contained the indicated number of mitochondria (1- >6). 150 DMSO axons and 105 CB-D axons from two rats were included. (H) The bar graph shows the percentage with 0, 1, 2 or >2 mitochondria for each DMSO treated nerve and each CB-D treated nerve.

Fig 4: SNX6 is required for motility of Homer1b/c vesicles in dendritic shaft and their association with dynein−dynactin.(A–C) Dynamic behavior of mCherry-SNX6 and mEmerald-Homer1c in distal dendrite. The last frame of motile SNX6-, Homer1c-positive puncta (arrowhead) in dendrite (A) with the respective kymograph of boxed area (B) is shown. A retrospective staining of MAP2 after live imaging (C) illuminates dendrite identity. (D) Superresolution images of DIV18 neurons immunostained with antibodies to SNX6, Homer1b/c, and p150Glued or DIC. Shown are representative images of dendrites outlined with dashed lines. White arrowheads indicate overlapped signals. (E) DIV14 neurons were transfected with construct expressing EGFP, treated with DMSO or Ciliobrevin D on DIV16 for 2 hr and immunostained with antibodies to Homer1b/c. (F) Quantification of puncta number per 100 μm dendrite length and mean intensity in spines for Homer1b/c in (E) (mean ± SEM, n = 30, N = 3). (G) Same as (E), shown are representative confocal images of EGFP-expressing neurons. Dashed lines outline the cell bodies. (H) Quantification of Homer1b/c distribution in the cell body and dendrites in (G) (mean ± SEM, n = 30, N = 3). (I) DIV14 neurons were transfected with construct overexpressing EGFP or p150Glued-N-EGFP, fixed on DIV16 and immunostained with antibodies to Homer1b/c. Dashed lines outline the cell bodies. (J) Quantification of Homer1b/c distribution in the cell body and dendrites in (I) (mean ± SEM, n = 30, N = 3). (K) Membrane fractions from mouse brain lysates were subjected to immunoisolation with antibodies to p150Glued or DIC coupled to Dynabeads Protein G, respectively. Shown are immunoblots probed with antibodies to SNX6, p150Glued, DIC, and Homer1b/c. (L) Hippocampal neurons cultured from Snx6fl/fl and Nestin-Cre; Snx6fl/fl mice were transfected with pLL3.7.1 on DIV14, fixed on DIV17 and immunostained with antibodies to Homer1b/c and p150Glued, DIC or EEA1. Shown are representative confocal images of dendritic segments. (M) Quantification of colocalization in (L) from 45 dendritic segments of 15 neurons (mean ± SEM, N = 2. Total length of dendrites: Snx6fl/fl/Nestin-Cre; Snx6fl/fl: 1247 μm/1058 μm for p150Glued; 1264 μm/1301 μm for DIC; 1244 μm/1291 μm for EEA1). Bars, 2 μm in (A), (D), (E) and (L), 20 μm in (G) and (I).DOI: http://dx.doi.org/10.7554/eLife.20991.014

Fig 5: The integrity and sucrose sedimentation pattern of the dynein complex is not compromised in SOD1G93A and SOD1G93A/Loa, but the Loa mutation in dynein alters the sedimentation of mutant SOD1 in SOD1G93A/Loa mice. A, brain homogenates of equal protein contents prepared from +/+, Loa/+; SOD1G93A, and SOD1G93A/Loa mice (at end stage of disease in the SOD1G93A and SOD1G93A/Loa mice) were sedimented in 5–40% continuous sucrose gradient by centrifugation at 237,000 × g for 4 h at 4 °C as described under “Experimental Procedures.” Then 16 equal-volume fractions. were collected from top to bottom and analyzed by SDS-PAGE and Western blotting detection for DHC, p150, DIC, and dynein light intermediate chain (DLIC) as representatives of the dynein-dynactin complex. B, shown are brain homogenates of equal protein contents prepared from SOD1G93A and SOD1G93A/Loa littermates at the postnatal stages; P35 (before disease onset), P75 (onset of disease), P90 (early stage of disease), P120 (late stage of disease), and end-stage, were sedimented in 5–40% continuous sucrose gradient by centrifugation at 237,000 × g for 4 h at 4 °C as described under “Experimental Procedures.” Then 16 equal-volume fractions were collected from top to bottom and analyzed by SDS-PAGE and Western blotting detection for SOD1 (to avoid overexposure caused by much higher levels of mutant SOD1 (mSOD1) protein in the lighter fractions of the gradient, we loaded 3 μl of fractions 1–3, 8 μl of fractions 5–7, 10 μl of fractions 8–9, and 20 μl of fractions 11–16; supplemental Fig. 3 shows the true representations of equal loading of the fractions on SDS-PAGE immunoblots). C, -fold difference (n = 3–6) in mutant SOD1 protein in fraction 16 of the SOD1G93A mice was quantified relative to that in SOD1G93A/Loa mice using COX4 as the internal control.