The following genotypes were used:
w - w; TBPHΔ23/Cyo-GFP  - w; TBPHΔ142/Cyo-GFP  - w; Mef2-GAL4/TM3-Sb - w; MHC-GAL4/TM3-Sb - w; UAS-mCD8::GFP/Cyo - w; UAS-TBPH/Cyo - w; UAS-TBPHF/L/TM3-Sb - w; UAS-hTDP/TM3-Sb - w; UAS-DLG(egfp)/UAS-DLG(egfp) - w; UAS-TBPH RNAi (#ID38377)  - w; UAS-GFP RNAi (#9330) - w; UAS-GFP RNAi (#9331) - w, Dicer(X).
Wandering 3rd instar larvae (about 96 h old) were picked from tubes and washed in a drop of demineralized water. If necessary, they were selected against different markers such as tubby or Cyo-GFP and placed into 6-cm-diameter dishes, filled with 0.7% agar. A single larva at the time was transferred into a 10-cm-diameter dish, filled with 0.7% agar. After 30 s of adaption period, the number of peristaltic waves were counted for a period of 2 min. Tested larvae were subsequently transferred to a fresh fly tube to check them, both for hatching (after 4 days) and for correct genotype selection. Generally, 20–25 larvae per genotype were tested.
One- to 2-day-old adult flies were collected from the fly tube of the experimental cross in a 1:1 proportion of female and male and transferred to a fresh fly tube and stored in the incubator under controlled conditions (suitable temperature and humidity, 12 h light and 12 h night). Every second day, flies were transferred into a fresh fly tube without anesthesia and the number of deaths was scored. Approximately 200 flies per genotype were tested.
One- to 2-day-old adult flies were collected from the fly tube of the experimental cross in a 1:1 proportion of female and male and transferred into a fresh fly tube and maintained in an incubator as previously described. The day of the setting of the experiment was counted as day 0. Flies were tested on days 4, 7, 14, and 21. A 50-ml glass cylinder was divided into three parts, as bottom, middle, and top (5 cm each part). Flies were carefully flipped into the cylinder from the fly tube without any anesthesia and gently dropped to the bottom. After 30 s of adaptation period, flies were dropped again onto the bottom of the cylinder, and after the time interval of 15 s, the numbers of flies present in each part of the cylinder were scored. For each genotype, 3 trials per tube were done and the average of the scored fly numbers was considered as the final score. A minimum of 200 flies was tested for each genotype.
Young flies 2–3 days old were tested for walking ability. A 145-mm dish was used. The bottom surface was divided in a grid of 1 cm × 1 cm squares to facilitate the measuring of the distance walked by flies. The fly without any anesthesia was placed in the middle of the dish, and after 30 s of adaptation to the environment, the distance walked by the fly was recorded for 30 s, counting the number of squares. A minimum of 50 flies were individually tested for each genotype.
Wandering 3rd instar larvae were picked from the fly tube, in a drop of demineralized water, selected for the genotype, and maintained during the time of dissection in a 6-cm-diameter dish filled with 0.7% agarose dissolved in water. Individually picked larva was dissected on Sylgard plates, in Dissection Solution (128 mM NaCl, 2 mM KCl, 4 mM MgCl2, 0.1 mM CaCl2, 35.5 mM sucrose, and 5 mM Hepes (pH 7.2)). Larvae were pinned at both ends with minute pins (Austerlic Isect Pins 0.1 mm diameter, Fine Science Tools, Germany) and opened on the dorsal site with Spring scissors (Fine Science Tools, Germany). Once larva was opened, internal organs were removed and the interior was extensively washed with Dissection Solution leaving the muscle wall opened, pinned flat on the surface. The subsequent step was a fixation, generally done with 4% PFA in PBS for 20 min; however, in the case of glutamate receptors staining a methanol fixation of 5 min at − 20 °C was performed. Fixation solution was removed with 3 washes in PBS-T (PBS 1× supplemented with 0.1% (v/v) Tween20) for 5 min each. After a blocking step of 30 min in blocking solution (5% NGS (normal goat serum (#S-1000 Chemicon) in PBS-T buffer), larvae were incubated overnight at 4 °C in primary antibodies diluted in blocking solution. The day after, primary antibody was removed with three washes of 10 min each with PBS-T and a further blocking step of 30 min was performed before secondary antibody addition. All secondary antibodies were diluted in blocking solution. An incubation was 2 h long, carried out at the room temperature. Excess of antibody was removed by 3 subsequent washes of 20 min each in PBS-T. Finally, dissected-stained larvae were incubated overnight at 4 °C in Slowfade®Gold antifade (#S36936 Life Technologies) reagent, before being mounted on a glass slide: anti-HRP (#323-005-021, lot:104838, Jackson 1:150), anti-GFP (#A11122, lot:1789911, Life Technologies 1:200), anti-GluRIIA 8B4D2c (DSHB 1:15), anti-Dlg 4F3c (DSHB 1:250), anti-Futsch 22C10s (DSHB 1:50), Alexa-Fluor® 488 (mouse #A11001 or rabbit #A11008 1:500), and Alexa-Fluor® 555 (mouse #A21422 or rabbit #A21428 1:500).
Acquisition and quantification of confocal images
In each experiment, the genotypes of interest were processed simultaneously, and the images were acquired using the same settings. Images of muscles 6 and 7 on second abdominal segment were acquired using the LSM Zeiss Software on a Zeiss 510Meta confocal microscope (63 × oil lens) and then analyzed using ImageJ (Wayne Rasband, NIH). For the quantification of pre- and postsynaptic markers, samples were double labeled with anti-HRP and the marker of interest: the ratio between the mean intensity of the marker and the HRP was calculated for each bouton of the terminal [18, 19].
Quantification of boutons
Boutons were stained with an anti-HRP antibody. The shape of boutons was evaluated as regular if they were round and with a smooth surface, with an equal diameter on both axes. On the other hand, boutons were considered as irregular if the shape was not round, the membrane was wrinkled, and the diameter of one axe was different compared to the other one .
Electrophysiology on NMJ of the third instar larva preparation
Larval body wall preparations were dissected out in Ca2+-free HL3 solution from third instar larvae pinned on Sylgard-coated petri dishes. The central nervous system was excised by cutting segmental nerve roots. After replacing Ca2+-free HL3 solution with Ca2+ 1 mM HL3, postsynaptic potentials at neuromuscular junction of fiber 6/7 of abdominal segments A3/A4 were intracellularly recorded, at room temperature in current-clamp condition, using an intracellular microelectrode (tip diameter 0.5 μm, 15 MΩ resistance). The recorded signal was amplified by a current-clamp amplifier (SEC 05, NPI, Tamm, Germany), digitized at 10-kHz sampling rate using an A/D interface (National Instruments, Austin, TX, USA) and fed to a computer for display and storage using an appropriate software (Win EDR, Strathclyde University, Glasgow, UK).
Fibers with a resting membrane potential below − 60 mV were considered for the experiment. In these fibers, membrane potential was set at − 70 mV throughout the experiment by injecting current through the intracellular electrode. Evoked postsynaptic potentials (EPSPs or excitatory junctional potentials or EJPs) were recorded by stimulating at 0.1 Hz (pulse duration 0.4 ms; 1.5 threshold voltage) the segmental nerve using a suction electrode (tip diameter ~ 10 μm) connected to a stimulator (S88, Grass, Pleasanton, CA, USA) through a stimulus isolation unit (SIU5, Grass, Pleasanton, CA, USA). Intracellular recordings were analyzed offline using pClamp software (pClamp, Axon, Sunnyvale, CA, USA). Statistical comparisons and graphs were made using Graphpad software (Graphpad, La Jolla, CA, USA) or MATLAB (Matworks, Natick, MA, USA).
Protein G magnetic beads (#10003D Invitrogen) were washed two times with PBS + 0.02% Tween and coated with anti-FLAG M2 monoclonal antibody (#F3165, Lot:SLBQ7119V, Sigma). Thoraces or heads of adult flies were cut and stored in lysis buffer containing 20 mM Hepes, 150 mM NaCl, 0.5 mM EDTA, 10% Glycerol, 0.1% Triton X-100, 1 mM DTT, and protease inhibitor (#04 693 159 001 Roche). Samples were homogenized with a Dounce homogenizer, and major debris were removed by centrifugation step of 5 min at 0.4g at 8 °C. The pretreated beads and tissue extracts were mixed and incubated for 30 min at 4 °C. After this binding step, beads were washed five times with washing buffer (20 mM Hepes, 150 mM NaCl, 0.5 mM EDTA, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, protease inhibitor, 0.2% DOC, 0.5 M Urea) using DynaMagTM-Spin (#123.20D Invitrogen). RNA transcripts bound by Flag-tagged TBPH were extracted treating the beads with Trizol (#15596026 Ambion), and RNA was precipitated with isopropanol adding glycogen (#R0551 Thermo Scientific). Retro-transcription was performed with Superscript III First-Strand Synthesis (#18080-093 Invitrogen) and oligo-dT and subjected to real-time PCR with gene-specific primers, whose sequences are listed below.
In order to calculate the enrichment fold, initially, all data were normalized to the respective inputs. The signal was represented by how many more fold increase was measured compared to the control signal. The enrichment was calculated according to the 2-ΔΔCt method.
The results were derived from three independent immunoprecipitation experiments [19, 20].
To collect adult heads, flies were flash-frozen in liquid nitrogen for 10 s and immediately vortexed to easily detach heads from bodies. Heads were subsequently transferred into Lysis buffer (150 mM Tris, 5 mM EDTA, 10% glycerol, 5 mM EGTA, 50 mM NaF, 4 M urea, 5 mM DTT, and protease inhibitors (#04 693 159 001 Roche)). After a squeezing step, performed both, manually and mechanically, the homogenized samples were gotten rid of major debris by centrifugation at 0.5×g for 6 min on 4 °C. The protein concentration of the collected supernatant was quantified with Quant-iT™ Protein Assay Kit (#Q33212 Invitrogen), following the supplier protocol.
Transfected neuroblastoma cell line SH-SY-5Y was resuspended in iced RIPA buffer added of protease inhibitors (#04693159001 Roche) and subjected to sonication (Biorupture sonication system, Diagenode).
Lysates were quantified (BCA Protein kit #23225 Thermo Scientific), following the supplier protocol.
RNA extraction and qRT-PCR
RNA was extracted from Drosophila adult heads, 1 day aged and sex-matched, of both wildtype and TBPH-null alleles (tbphD23 and tbphD142) and from human MN differentiated cells with RNeasy Microarray tissue kit (QIAGEN #73304) and treated with Turbo DNA-free kit (Ambion #AM1907).
Retro-transcription was performed with Superscript III First-Strand Synthesis (#18080-093 Invitrogen) using oligo-dT with the exception of the analysis of the Drosophila intronic region for which random hexamers have been used. Real-time PCR was carried out with the primers listed below using Platinum Syber Green (#11733-038 Thermo Fisher) on a Bio-Rad CFX96 qPCR System, and minus-retro control has been performed. The primer sets used are listed in the below table; Sdha and GAPDH genes have been used as Drosophila and human reference, respectively.
d-DLG exon 1–4
d-DLG intron 1–4
Protein samples were diluted in 1x Laemmli buffer (composition of 5x: 0.3 M Tris-HCl pH 6.8, 50% glycerol, 10% SDS, 25% β-mercaptoethanol, 0.05% bromophenol blue) to reach the same concentration among all and then boiled at 95 °C for 5 min. Afterwards, they were loaded on a polyacrylamide gel.
The loaded gel was placed into a chamber with 1× running buffer (10× running buffer: 30.28 g Tris, 114.13 g glycine, 10 g SDS in 1 l water). The conditions set were 25 mA per gel.
When proteins were separated by the electrophoresis, they were transferred to a nitrocellulose membrane Amersham™ Protran™ 0.2 μm NC (Life Science). The western blot sandwich was put into the chamber, filled with transfer buffer 1× containing 20% methanol (transfer buffer 10×: 30 g Tris, 144 g glycine in 1 l water). The transfer lasted 1 h at 350 mA. The membrane was incubated with a solution of 5% milk in 1× TBS 0.01% Tween (TBS-T) for 30 min at a room temperature on a shaker (TBS buffer 10×: 24.2 g Tris, 80 g NaCl in 1 l water, pH 7.6). After blocking, the membrane was set into dilution of primary antibody with TBS-T with 5% milk. It was placed at 4 °C overnight. When the incubation with primary antibody was over, five washes with TBS-T followed, 5 min each. Next, the membrane was incubated with the secondary antibody diluted in TBS-T with 5% milk for 1 h at room temperature. The protein detection was performed with SuperSignal®West Femto Maximum Sensitivity Substrate (#TB260893 Thermo Fisher Scientific). Primary antibodies include anti-Dlg 4F3c (DSHB 1:10000), anti-TBPH (home-made 1:4000), anti-tubulin (#CP06, Lot:2681308, Calbiochem 1:4000), anti-TDP-43 (#12892-1-AP, Lot:00016371, Proteintech 1:4000), anti-Dlg 2D11 (#SC9961, Lot:G2617, Santa Cruz 1:1000), and anti-GAPDH (#SC25778, Lot:G1008, Santa Cruz 1:2000). Secondary antibodies include anti-mouse-HRP (#31430 Thermo Fisher Scientific 1:30000(flies), 1:10000(cells)) and anti-rabbit-HRP (#31460 Thermo Fisher Scientific 1:10000).
Cell culture and RNA interference
SH-SY-5Y neuroblastoma cell line was cultured in standard conditions in DMEM-Glutamax (#31966-021, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum and 1 × antibiotic-antimycotic solution (#A5955; Sigma). RNA interference of TDP-43 was achieved using HiPerfect Transfection Reagent (#301705, Qiagen) and siRNA specific for human TDP43 (5′-gcaaagccaagaugagccu-3′); as control, siRNA for luciferase was used (5′-uaaggcuaugaagagauac-3′; Sigma). Immediately before transfection, 2–4 × 105 cells were seeded in 6-well plates in 1.4 ml of medium containing 10% fetal serum. A volume of 3 μl of each siRNA (40 μM solution in water) was added to 91 μl of Opti-MEM I reduced serum medium (#51985-026, Thermo Fisher Scientific) and incubated 5 min at room temperature, and subsequently, 6 μl of HiPerfect Transfection Reagent was added. The silencing procedure was performed again after 24 and 48 h.
Human iPSC culture and MN differentiation
Human iPSC culture and MN differentiation were already described in . All the studies performed with human samples were in compliance with the Code of Ethics of the World Medical association (Declaration of Helsinki) and with the national legislation and institutional guidelines. Briefly, fibroblasts from dermal biopsies (Eurobiobank) from ALS patients (TDP-43 mutations #1:G294V; #2:G378S) and controls were reprogrammed into iPSCs with CytoTune-iPS 2.0 Sendai reprogramming Kit (#A16517, Thermo Fisher) and differentiated into MNs with the multistep protocol described by Ng .
All statistical analysis was performed with Prism (GraphPad, USA) version 5.1. One-way ANOVA with Bonferroni correction and t test with Mann-Whitney correction were applied as statistical test. In all figures, all the values were presented as the mean and the standard error of the mean (SEM). Statistical significance was portrayed as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.