As described previously [37, 42], rNLS8 mice were generated by crossing animals expressing the tetracycline transactivator (tTA) protein under the control of the human NEFH promoter with a second line transgenic for human TDP-43 with a defective nuclear localization signal (hTDP-43ΔNLS) under a “tetracycline-off” promoter. In the resulting rNLS8 bigenic mice, dietary doxycycline (Dox Diet #3888, Bio-Serv) inhibits tTA from binding to the tetracycline promoter element, thereby suppressing hTDP-43ΔNLS. Transgene expression was induced by substituting standard chow (Rodent Diet 20 #5053, PicoLab). Male and female rNLS8 mice were randomized to treatment groups at age 2–5 months. Investigators were blinded to the genotype and treatment of each mouse during data collection, and identities were unblinded subsequently for analysis using tattooed identification numbers. Sample sizes were chosen based on power estimates informed by prior publications using the rNLS8 model [37, 40]. All procedures observed the NIH Guide for the Care and Use of Experimental Animals. Studies were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
To label MNs innervating the tibialis anterior (TA) with GFP before disease onset, P4 pups (n = 16) were anesthetized by hypothermia and the TA muscle was injected with 1 μL of AAV9-CMV-PI-eGFP-WPRE-bGH vector (3.3 × 1013 gc/mL, Penn Vector Core) using 31-gauge needles with 1/2 cc insulin syringes (BD Ultra-Fine∗ II Short Needle Insulin Syringe, VWR). Pups were returned to their mother and their health was monitored to adulthood. Upon reaching adulthood, the mice were randomized between hTDP-43-ΔNLS insult (Dox withdrawn, n = 8) and control (maintained on Dox for transgene suppression; n = 8) cohorts. After 6 weeks of hTDP-43-ΔNLS expression, doxycycline was reintroduced to allow axonal regeneration to occur. After 8 weeks of recovery, Cholera Toxin Subunit B conjugated to Alexa Fluor 594 (CTB594; 3 µL, Invitrogen) was injected into the same TA muscle. All animals were sacrificed 4 days thereafter, and the lumbar 3–4 regions of the SC were analyzed for tracer labeling of the neuronal soma.
CTB-594 (3 µL) was injected into the TA of mice as described above to effect retrograde labelling in the L3 level of the lumbar spinal cord of MNs innervating the injected muscle . Four days after CTB-594 administration, the mice were perfused with PBS followed by 4% paraformaldehyde and the lumbar SC removed for analysis of the TA motor pool.
To perform compound muscle action potential (CMAP) recordings, mice were anesthetized using a KAX cocktail (ketamine 60–100 mg/kg, xylazine 8–12 mg/kg, acepromazine 0.5–2 mg/kg), and their hind legs were shaved. The sciatic nerve was stimulated with brief electrical currents applied using bipolar needle electrodes (0.3 Hz, 0.5-ms pulse duration, starting at 0 mA and incrementing by 5 mA). The response from the gastrocnemius (GC) muscle was recorded using needle electrodes placed in the center of the muscle and in the tendon . The M-wave was measured at each amplitude until the maximal response was elicited. Maximum evoked amplitudes were analyzed using the pCLAMP 10 software suite (Molecular Devices).
The mice were anesthetized as described above, the lateral thigh of rNLS8 or non-transgenic (nTg) mice was shaved, and a 0.5–1 cm incision in the skin was made over the lateral femur. For the nerve crush, the sciatic nerve was located and crushed with a hemostat for 15 s . The skin incision was closed with silk sutures, and the animals were allowed to recover on a warming blanket.
Surgery was performed aseptically following the IACUC Guidelines for rodent survival surgery. The mice were anesthetized as described above, the lateral hindquarters at the incision site were shaved, and a 0.7–1 cm cutaneous incision was made. The fascial plane was opened between the gluteus maximus and the anterior head of the biceps femoris to reveal the common peroneal and tibial nerves. For cross-reinnervation surgery, the nerves were severed, and the proximal stump of the tibial nerve was cross-sutured to the distal stump of the common peroneal nerve via a 3 mm silicon laboratory tubing (0.025 in ID, 0.047 in OD, Fisher Scientific) with 10.0 nylon. The distal stump of the tibial nerve was similarly cross sutured to the proximal stump of the common peroneal nerve. For surgical control, the nerves were exposed and severed, and the proximal stump of the common peroneal and tibial nerves was sutured to the distal stump of corresponding the common peroneal and tibial nerves via a 3 mm silicon laboratory tubing with 10.0 nylon for self-reinnervation surgery. The gluteal musculature was then re-opposed and sutured with 6.0 nylon skin incision. After surgery, mice were maintained under a warming lamp until fully conscious.
rNLS8 and nTg mice were perfused with ice-cold PBS followed by 4% paraformaldehyde, and the brain, lumbar SC, and hindlimb muscles were collected. Peripheral tissues were washed in PBS overnight, while CNS tissues were fixed in 10% formalin overnight. All tissues were then washed in PBS and processed in a sucrose gradient up to 30% for cryoprotective embedding. To analyze MN populations in the brainstem and lumbar SC, tissues were sectioned at 20 μm thickness, incubated with citric acid (95 °C for 3 min) for antigen retrieval, incubated in 5% FBS in PBS for blocking and immunostained using the following primary antibodies: guinea pig anti-VAChT (1:1000, CNDR, U. Pennsylvania), rabbit anti-VAChT (1:5000, CNDR, U. Pennsylvania), mouse anti-SV2 (2–5 µg/mL, DSHB, U. Iowa), rabbit anti-NFL (1:500, CNDR, U. Pennsylvania), mouse anti-human TDP-43 mAb (0.06 μg/mL, CNDR, U. Pennsylvania, clone 5104), rabbit anti-MMP9 (1:1000, Abcam), goat anti-MMP9 (1:2000, Sigma-Aldrich), rabbit anti-SK3 (Kca2.3, KCa3, Kcnn3, SKCa3) polyclonal (1:500, Millipore), rat anti-phospho-TDP-43S409−S410 (1:5000, Cosmo Bio Co.), and mouse anti-pan-TDP-43 (1:10.000, CNDR, U. Pennsylvania). After overnight incubation, tissue sections were washed and then incubated with AlexaFluor secondary antibodies (1:1000, Molecular Probes) for 2 h. Sections were imaged using either a Nikon Eclipse Ni inverted microscope or a Leica TCS SPE confocal microscope. For confocal imaging, 10 z-steps spaced 1–3 µm apart were collected per image, and a maximum projection was created for each. For the quantification, MNs were counted in transverse 20 µm cryosections, 100 µm apart, over a length of 1 mm, using Image J and NIS-Elements software.
The soleus, lateral GC and TA muscles were dissected [13, 32] and sectioned at 30 μm longitudinally, and immunostained for VAChT or SV2 (as described above) to label nerve terminals and treated with α-bungarotoxin conjugated to Alexa Fluor 488 (BTX, 1:1000) to label motor endplates. The proportion of innervated NMJs was scored as the number of areas positive for both SV2 (or VAChT) and BTX (i.e., the total number of innervated endplates) divided by the total number of all BTX-positive areas (i.e., the total number of endplates) on consecutive longitudinal 30 μm cryosections. In this manner, 500–1000 NMJs were scored per TA and lateral GC, and 200–500 NMJs per soleus.
Muscle fiber typing
rNLS8 and nTg mice were perfused with ice-cold PBS followed by 10% (v/v) neutral buffered formalin. The hindlimb muscles were collected, washed in PBS overnight and processed in a 10–30% sucrose gradient for cryoprotective embedding. The muscles were cross-sectioned at 10 µm thickness, incubated in 5% FBS in PBS for blocking, and then immunostained using the following primary antibodies: mouse anti-BA-D5 (for myosin heavy chain type I; 2–5 µg/mL, DSHB, U. Iowa), mouse anti-BF-F3 (for myosin heavy chain type IIB; 2–5 µg/mL, DSHB, U. Iowa), mouse anti-SC-71 (for myosin heavy chain type IIA; 2–5 µg/mL, DSHB, U. Iowa) . After overnight incubation, tissue sections were washed and then incubated with Alexa Fluor secondary antibodies (1:1000, Molecular Probes) for 2 h. Sections were imaged using a Nikon Eclipse Ni inverted microscope.
Gait analysis was performed using the TreadScan software from the CleverSys NeurodegenScan Suite. The BcamCap image capture system recorded the footprints of the mice locomoting across a transparent treadmill using a high-speed camera at 100 frames per second. The mice were trained on the treadmill at 10–20 cm/s for 20 s periods for 5 training sessions over a 10-d period. The chamber and treadmill were wiped down with 0.25% bleach then with distilled water. After a 3-min habituation on the treadmill, the track speed was slowly increased from 5.0 cm/s until the mouse could no longer keep pace or until 20.0 cm/s was achieved [37, 42]. Instantaneous running speeds and running times were derived from image analysis by the TreadScan software and output for statistical analysis.
Data were first tested for normality (Shapiro–Wilk test) and equivalent variances (Brown-Forsythe test). To determine the statistical significance of comparisons between two groups, unpaired two-tailed t tests were used, except for nerve crush experiments, which used paired two-tailed t tests. One-way ANOVA with the Holm-Sidak method for pairwise multiple comparisons was used when comparing multiple groups. Two-way ANOVA using Tukey’s multiple comparison test was used to evaluate the effect of two factors (e.g., surgical procedure and time post-surgery). Statistical tests were implemented in SigmaPlot and GraphPad Prism 7. P-values < 0.05 were considered statistically significant.