PMC

TDP-43 is expressed in dynamic cytoplasmic puncta in primary motor neurons. (A–F) TDP-43 expressed using D42 Gal4, visualized with anti-GFP antibody, localizes to distinct puncta within neurites (arrows). Neuronal membranes were labeled with anti-HRP antibodies. (G) Velocity quantification of TDP-43-containing puncta (mean, maximum and minimum velocities). (H) Quantification of total and net distances for TDP-43 puncta. (I) FRAP indicates that TDP-43 is mobile within neurites. Note that wild-type kinetics are significantly different to those of mutant TDP-43 (see text for discussion). Values show means ± s.e.m. Scale bar: 15 μm.

TDP-43 variants affect the growth and function of the neuromuscular junction synapse. (A–F) Neuromuscular junctions in larvae expressing TDP-43 driven by D42 Gal4 were labeled by CSP (synaptic vesicle marker) and HRP (neuronal membrane marker). (G) Quantification of synaptic boutons indicates a reduction in synaptic size due to TDP-43 overexpression. (H) Larvae expressing TDP-43 in motor neurons are impaired in larval turning behavior. (I–N) Neuromuscular junctions in larvae expressing TDP-43 driven by repo Gal4 were labeled by CSP (synaptic vesicle marker) and HRP (neuronal membrane marker). (O) Quantification of synaptic boutons indicates a decrease in synaptic size due to TDP-43 overexpression. (P) Larvae expressing TDP-43 in glia are impaired in larval turning behavior. Values show means ± s.e.m. All comparisons were performed using D42 Gal4 driver controls and Student’s t-test was used to calculate significance; *P<0.5, **P<0.01, ***P<0.001. Scale bars: 30 μm.

Active zones and postsynaptic glutamate receptor distribution are differentially affected at the neuromuscular junction by TDP-43 expression in motor neurons versus glia. (A–F) The presynaptic marker Bruchpilot (visualized with NC82 antibodies) and postsynaptic marker glutamate receptor (GluR) were used to label neuromuscular junctions in larvae expressing TDP-43 in motor neurons (staining and genotype as indicated). (G,H) Quantification of NC82 puncta indicates a significant increase in the number of presynaptic active zones per bouton (G), which is not accompanied by a similar increase in postsynaptic GluR (except for wild-type TDP, see H). (I–N) The presynaptic marker Bruchpilot (visualized with NC82 antibodies) and postsynaptic marker GluR label neuromuscular junctions in larvae expressing TDP-43 in glia (staining and genotype as indicated). (O,P) Quantification of NC82 puncta shows no effect on the number of presynaptic active zones per bouton (O), but a significant increase in postsynaptic GluR expression (P). Values show means ± s.e.m. All comparisons were performed using Gal4 driver controls and Student’s t-test was used to calculate significance; *P<0.5, **P<0.01, ***P<0.001. Scale bars: 5 μm.

TDP-43 forms axonal aggregates in developing eyes. (A–H) Confocal images (single slices, 1 μm each or 2–3 slice projections) showing GFP NLS (A,E) or TDP-43 variant (B–D,F–H) localization when expressed in the developing eye neuroepithelium with GMR-Gal4. TDP-43 visualized via individual fluorescent YFP tags (indicated as GFP here). Filamentous actin labeled with phalloidin, DNA stained with Hoechst 33342 as indicated. Note TDP-43 puncta in axons (arrowheads in B–D,F–H and insets); compare with GFP NLS controls (arrowheads in A,E and inset). (I) Cellular fractionation of adult head tissue shows TDP-43 distribution complexes ranging from highly soluble (LS, low salt) to insoluble (U, urea). Triton X-100 (TX) and sarkosyl (SK) correspond to non-ionic and ionic detergent soluble fractions, respectively. TDP-43 was detected using anti-GFP antibodies. Tubulin was used as a loading control. Scale bar: 75 μm.

TDP-43 expression in motor neurons or glia leads to cytoplasmic aggregates and nuclear morphology defects. (A–F) TDP-43 expressed with D42 Gal4 was visualized using anti-GFP antibodies (B–F) and compared with D42 Gal4 >GFP NLS controls (A). Motor neurons within the ventral ganglia were labeled by GFP; DNA was stained with Hoechst 33342. (G–I) Mutant variants but not wild-type TDP-43 alter nuclear shape by increasing eccentricity (G), decreasing the form factor (H) and increasing compactness (I) compared with D42 Gal4 driver controls. (J–P) TDP-43 expressed in glia with repo Gal4 was visualized using anti-GFP antibodies (K–P) and compared with repo Gal4 >GFP NLS controls stained with Hoechst 33342 to visualize DNA (J). Note the presence of TDP-43 puncta in the cytoplasm (K–P). (Q–V) TDP-43 is expressed in puncta (arrows) within glial cells enveloping the neuromuscular junction synapse labeled by CSP and HRP. (W–Y) Glial expression causes relatively mild changes in nuclear shape, primarily in D169G and N345K compared with repo Gal4 driver controls. Eccentricity, form factor and compactness are shown as means ± s.e.m.; ***P<0.001. Scale bars: 20 μm.

Overexpression of mutant TDP-43 leads to age- and dose-dependent neurodegeneration in the adult retina. (A–L) GMR Gal4 expression of TDP-43 D169G (D–F), G298S (G–I) and N345K (J–L) variants results in age-dependent loss of pigment compared with GMR Gal4 driver controls (A–C) at 25°C. Note that D169G depigmentation is milder than in G298S and N345K variants. (M–X) TDP-43 variants D169G TDP-43 (P–R), G298S TDP-43 (S–U) and N345K TDP-43(V–X) exhibit stronger surface phenotypes, including necrosis, compared with controls (M–O) when expressed at higher levels (29°C). Genotypes and ages are indicated. Anterior right, dorsal up. (Y) Western blot analyses showing TDP-43 expression in several transgenic lines. For each mutant variant we characterized multiple lines, two of which are shown. Genotypes are indicated on top; blotting antibodies are indicated on the left. Arrowheads on the right indicate full-length and C-terminus truncation of TDP-43. Tubulin was used as a loading control.

TDP-43 overexpression affects sleep and locomotor activity in adult flies. (A–F) TDP-43 variants were expressed in motor neurons using D42 Gal4. Measurements using DAMs show that locomotor activity is significantly reduced overall (A) and total sleep is significantly altered, with wild-type TDP-43 leading to longer overall sleep than the mutant variants (B). During the day (C,D) as well as during the night (E,F), TDP-43 expression results in significantly more sleep episodes (D,F) that last shorter intervals of time (C,E). (G–L) TDP-43 variants were expressed in glial cells using repo Gal4. Measurements using DAMs show variable effects on locomotor activity (G) and a significant reduction in total sleep (H). During the day (I,J) there are variable effects on sleep. During the night (K,L), TDP-43 expression in glia results in significantly more sleep episodes (L) that last shorter amounts of time (K). Values show means ± s.e.m. All comparisons were performed using D42 Gal4 driver controls and significance was determined using a non-parametric Wilcox test calculated in R; *P<0.5, **P<0.01, ***P<0.001.