Article Figures & Data
Figures
- Figure 1.
Futsch mRNA associates with TDP-43 in a complex. A, RIP of TDP-43 variants in Drosophila heads. Genotypes and antibodies used for RIP are indicated at the top. The antibody used for Western blot analysis is indicated on the right. B, RT-PCR of immunoprecipitated TDP-43 complexes. Genotypes and antibodies used for IP as indicated in A. mRNA is indicated on the right. C, qPCR of futsch mRNA in immunoprecipitated TDP-43 complexes.
- Figure 2.
TDP-43 inhibits Futsch expression post-transcriptionally, and futsch mRNA localization and translation are inhibited in the context of TDP-43. A–D, Larval ventral ganglia of D42>w1118 controls (A, B) and larvae expressing TDP-43WT (C, D) in motor neurons immunostained for Futsch and Hoescht. E–H, Larval neuromuscular junctions of D42>w1118 controls (E, F) and larvae expressing TDP-43WT (G, H) in motor neurons immunostained for Futsch and the neuronal membrane marker HRP. I–K, qPCR of futsch mRNA in the context of TDP-43WT and TDP-43G298S overexpression versus controls in whole larvae (I), ventral ganglia (VG; J), or neuromuscular junctions (K). L, Quantification of Futsch protein intensity at the neuromuscular junction normalized to HRP and muscle areas. All images were acquired using identical parameters. M, N, Western blots for Futsch levels in VGs (M) and NMJs (N) of TDP-43-expressing larvae. Genotypes are indicated at the bottom. Actin was used as a loading control. O, P, Quantification of Futsch protein levels from Western blots represented as a ratio to D42>w1118 controls. Q, TDP-43WT and TDP-43G298S associate with RNPs and actively translating polysomes. R–T, qPCR of futsch mRNA in input (R), RNPs (S), and polysomes (T) in the context of TDP-43WT and TDP-43G298S compared with controls. **p < 0.05; ***p < 0.001, Student's t test. Scale bars: A, 20 μm; E, 55 μm.
- Figure 3.
Futsch is neuroprotective in the context of TDP-43 overexpression. A, D42 Gal4 expression of TDP-43 WT or G298S results in impaired larval turning behavior, which is not significantly altered by futsch RNAi but is rescued by futsch OE. B, C, D42 Gal4 expression of TDP-43 variants, WT (B) and G298S (C), results in reduced adult survival time, which is rescued by futsch OE. FutschRNAi shifts the lethality caused by TDP-43 to pupal stage (see Results). The Student's t test was used to calculate larval turning time p values. The log-rank test was used to calculate survival p values. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 4.
Futsch is neuroprotective at the Drosophila neuromuscular junction. A, C, E, Larval neuromuscular junctions of D42>w1118 controls (A) and larvae expressing TDP-43WT (C) or TDP-43G298S (E) in motor neurons immunostained for Futsch and the neuronal membrane marker HRP. B, D, F, Larval neuromuscular junctions of Futsch OE in a TDP-43WT (D) and TDP-43G298S (F) background compared with Futsch OE controls (B) immunostained for Futsch and the neuronal membrane marker HRP. All images were acquired using identical parameters. A1–F1, High-magnification views of HRP immunostaining shown in A–F. The white arrowhead or asterisk indicates bouton of interest. A2–F2, High-magnification views of Futsch immunostaining shown in A–F. White arrowheads indicate fully formed Futsch loops in the bouton of interest, and white asterisks indicate fuzzy Futsch staining in the bouton of interest. G, Quantification of bouton number normalized to muscle areas. H, Quantification of fuzzy Futsch loops normalized to HRP areas. ***p < 0.001, *p < 0.05, Student's t test. Scale bars: A, 35 μm; A1, 5 μm.
- Figure 5.
Futsch overexpression increases microtubule stability at the NMJ. A–C, Larval neuromuscular junctions of D42>w1118 controls (A) and larvae expressing TDP-43WT (B) or TDP-43G298S (C) in motor neurons immunostained for the neuronal membrane marker HRP. Arrowheads indicate satellite boutons. D–F, Larval neuromuscular junctions of Futsch OE in a TDP-43WT (E) and TDP-43G298S (F) background compared with Futsch OE controls (D) immunostained for the neuronal membrane marker HRP. Arrowheads indicate satellite boutons. G, Western blot analysis showing acetylated tubulin levels. Genotypes are indicated at the top. Antibodies are indicated on the left. Tubulin was used as a loading control. H, Quantification of satellite bouton number normalized to total boutons. I, Quantification of relative protein levels from Western blot analysis. *p < 0.05; **p < 0.01; ***p < 0.001, Student's t test. Scale bar, 5 μm.
- Figure 6.
TDP-43 aggregates are significantly decreased by Futsch OE. A, B, Solubility studies using third instar larvae show the distribution of TDP-43 variants WT (A) and G298S (B) in LS, Sarkosyl (Sark), and urea fractions alone in the context of Futsch overexpression (Futsch OE). C, D, Quantification of WT (C) or G298S (D) TDP-43 levels in LS, Sark, and urea fractions normalized to input. *p < 0.05; ***p < 0.001, Fisher's exact test.
- Figure 7.
MAP1B localization in ALS patient samples. A, B, MAP1B immunostaining in control (A) and ALS (B) lumbar spinal cords. Note the cell body accumulation of MAP1B in ALS tissues (compare arrowheads). C–J, MAP1B immunostainings in control (C, E, G, I) and ALS (D, F, H, J) dentate gyrus (DG) and CA1 regions of the hippocampus. Note the additional cell body and proximal neurite accumulation of MAP1B in the DG of the ALS/FTLD subject (F). Scale bars: A, 40 μm; C, 30 μm.
- Figure 8.
Model of futsch mRNA regulation by TDP-43. A, In controls, TDP-43 transports futsch mRNA to synapses where translation occurs, resulting in stabilized synaptic connections. B, In the ALS model, TDP-43 alters the localization of futsch mRNA, increasing its concentration in the cell body and reducing its concentration at the NMJ. Additionally, TDP-43 negatively regulates Futsch expression post-transcriptionally at the level of translation, resulting in decreased Futsch protein levels in both the cell body and at the NMJ relative to mRNA concentrations. Subsequently, this leads to decreased microtubule stability, increased aggregation of TDP-43, and reduced NMJ size, locomotor function, and life span. C, After Futsch overexpression in the context of TDP-43, futsch mRNA localization and Futsch protein levels are restored subsequently, resulting in a rescue of microtubule stability, TDP-43 solubility, as well as NMJ size, locomotor function, and life span.
Tables
D42>w1118 (absolute value; ratio to w1118 VG) D42>TDPWT (absolute value; ratio to w1118) D42>TDPG298S (absolute value; ratio to w1118) VG 1.20 ± 0.07; 1.0 1.96 ± 0.15; 1.63 (p = 0.01) 1.94 ± 0.16; 1.61 (p = 0.01) NMJ 1.72 ± 0.09; 1.4 0.92 ± 0.04; 0.53 (p = 0.001) 0.96 ± 0.06; 0.56 (p = 0.001) Futsch protein levels normalized to actin at the ventral ganglia (VG) and NMJ. Genotypes are as indicated. Mean ± SEM and p values are as shown. See also Fig. 8. Western blots were performed as described in Materials and Methods.
D42>w1118 D42>TDPWT (ratio to w1118) D42>TDPG298S (ratio to w1118) VG 1.0 3.99 3.15 NMJ 1.4 0.60 0.66 futsch mRNA levels at the ventral ganglia (VG) and NMJ in TDP-43-expressing larvae are represented as fold change compared with w1118 controls. w1118 futsch mRNA levels at the NMJ are represented as fold change compared with w1118 VG.
D42>w1118 D42>TDPWT D42>TDPG298S Futsch 5/6 1.0 1.02 1.12 Futsch 6/7 1.0 0.97 1.02 Futsch 8/9 1.0 1.03 0.92 futsch mRNA levels in whole larvae expressing TDP-43 using three different primer sets spanning different exon–exon junctions along the futsch transcript.
TDP-43WT (absolute value; ratio to input) TDP-43G298S (absolute value; ratio to input) Input 1.04 ± 0.02 1.15 ± 0.12 RNP 1.05 ± 0.02; 1.01 ± 0.001 1.14 ± 0.12; 0.99 ± 0.003 40S/60S 0.97 ± 0.06; 0.93 ± 0.04 1.05 ± 0.05; 0.93 ± 0.07 80S (monosome) 0.83 ± 0.05; 0.80 ± 0.03 0.88 ± 0.03; 0.78 ± 0.05 2 ribosomes 0.90 ± 0.04; 0.87 ± 0.02 0.85 ± 0.01; 0.75 ± 0.07 3 ribosomes 0.85 ± 0.04; 0.82 ± 0.03 0.85 ± 0.03; 0.76 ± 0.08 4 ribosomes 0.84 ± 0.05; 0.80 ± 0.03 0.84 ± 0.08; 0.74 ± 0.04 5 ribosomes 0.85 ± 0.03; 0.82 ± 0.02 0.83 ± 0.08; 0.73 ± 0.04 6 ribosomes 0.84 ± 0.01; 0.81 ± 0.01 0.82 ± 0.08; 0.72 ± 0.07 Quantification of TDP-43 protein distribution throughout the polysome gradient relative to S6 ribosomal protein distribution.
- Table 5.
Human tissues and quantification of MAP1B in the cytoplasm of spinal cord motor neurons
Case Diagnosis Age (years)/gender PMT (hours) Number of MNs counted Percentage of MNs with MAP1B in cell body C1 Control brain–no pathology 54/M 6 148 0 C2 Subacute infarcts 53/F 4 162 1.8 C3 Metastatic carcinoma 51/F 5 65 0 C4 Metastatic carcinoma 57/F 11 28 0 C5 Pulmonary fibrosis 57/M 2 108 0 C6 Control brain–no pathology 58/F 5 62 0 ALS1 ALS 62/F 4 32 19 ALS2 ALS 50/F 7 10 30.0 ALS3 Familial ALS (C9ORF72) 63/F 4 35 0 ALS4 ALS 60/F 4 44 66 ALS5 ALS 59/M 4 121 72 ALS6 ALS 63/F 2 51 59 ALS7 ALS 53/M 8 25 52 ALS8 ALS 55/M 3 37 57 PMT, Postmortem interval; MN, motor neuron; M, male; F, female. The percentage of spinal cord motor neurons containing MAP1B in the cell body was determined in motor neurons observed in between two and four tissue sections per case. The average percentage of motor neurons containing MAP1B in the cell body was 0.3 ± 0.3% per control case and 44 ± 8.9% per ALS case (±SD).
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