Animals

Mice were housed in the animal facility of the Institute for Clinical Neurobiology in compliance with national federal law and the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. As SMA mouse model, we used the established C57Bl/6 N/Smn1tm1Hung Tg(SMN2)2Hung/J line with C57BL/6J background [20]. SMA litters (Smn−/−,Hungtg/+, or Smn KO), and control litters (Smn+/−,Hungtg/+ or control) were derived from cross-breeding of Smn+/− to Smn−/−,Hungtg/tg. C57BL/6J mice were used as WT for control experiments. Munc13-1 KO mice (Munc13-1−/−), and Munc13-2 KO mice (Munc13-2−/−) [16, 18] were obtained from Nils Brose from Goettingen, Germany and cross-bred in-house.

Enrichment and culturing of primary mouse motoneurons

Primary mouse motoneurons were enriched via p75NTR antibody panning and cultured as previously described [8, 21, 22]. For lentivirus transduction, cell suspensions were briefly exposed to lentiviral particles for 10 min at RT and then plated on pre-coated polyornithine and laminin211/221 (Biolamina, LN211-0501, and LN221-0501) dishes. This specific laminin isoform has been shown to promote axonal growth cone differentiation into presynaptic structures in cultured motoneurons [10, 23]. Cells were grown onto glass coverslips for immunofluorescence, 24-well plates for Western blot and qRT-PCR, and µ-dishes (Ibidi, 81156) for Ca2+ imaging. Motoneuron culturing into compartmentalized microfluidic chambers was conducted as described earlier [24].

Immunocytochemistry

Cells were washed twice with pre-warmed PBS and fixed with 4% Paraformaldehyde (PFA) (ThermoFisher Scientific, 28908) for 10 min at RT and permeabilized with 0.1% Triton X-100. Block solution (2% BSA, 100 µg/ml saponin, and 0.25% sucrose in PBS) was added and incubated for 1 h at RT. Primary antibodies were diluted in block solution and incubated at 4 °C overnight. Primary antibodies were washed trice with TBST followed by secondary antibody incubation (diluted 1:500 in PBS) for 1 h at RT. Coverslips were embedded in Aqua Poly/Mount (Polysciences, 18606-20). For β-actin (Actβ) immunostaining, cells were permeabilized with ice-cold methanol for 5 min at −20 °C followed by a 5 min incubation at RT with 0.1% Triton X-100. Primary antibodies are as followed: rabbit polyclonal anti-Tau (Sigma-Aldrich, T6402, 1:1000), mouse monoclonal anti-α-Tubulin (Sigma-Aldrich, T5168, 1:1000), mouse monoclonal purified IgG anti-Basoon (Synaptic Systems, 141011, 1:500), guinea pig polyclonal antiserum anti-Piccolo (Synaptic systems, 142104, 1:500), rabbit polyclonal purified anti-RIM1/2 (Synaptic Systems, 140213, 1:500), rabbit polyclonal anti-Munc13-1 (Synaptic System, 126103, 1:500), rabbit polyclonal anti-Munc13-2 (Synaptic System, 126203, 1:400), rabbit polyclonal anti-Liprinα1 (Merck Millipore, ABT268, 1:500), guinea pig polyclonal purified anti-Ca2+ channel N-type alpha-1B (Cav2.2) (Synaptic System, 152305, 1:250), mouse monoclonal anti-SMN (BD Biosciences, 610647, 1:5000), and mouse monoclonal anti-β-actin (GeneTex, GTX26276, 1:1000). Secondary antibodies are as followed: donkey anti-mouse IgG (H + L) (Alexa Fluor 488, Jackson ImmunoResearch, 715-545-150), donkey anti-rabbit IgG (H + L) AffiniPure (Alexa Fluor 488, Jackson ImmunoResearch, 711-545-152), donkey anti-rabbit IgG (H + L) AffiniPure (Cy3, Jackson ImmunoResearch, 711-165-152), and donkey anti-guinea pig IgG (H + L) AffiniPure (Cy5, Jackson ImmunoResearch, 706-175-148).

Single-molecule fluorescence in situ hybridization (smFISH)

smFISH was conducted following the manufacturer’s instructions (ThermoFisher Scientific), as described earlier [8]. Briefly, after 10 min fixation at RT with paraformaldehyde lysine phosphate (PLP) buffer (4% PFA, 5.4% glucose, and 10 mM sodium metaperiodate, pH 7.4), cells were permeabilized for 4 min at RT with a supplied detergent solution. Cells were treated with proteinase K (diluted 1:8000 in PBS) for 4 min at RT. Hybridization probes (diluted 1:100 in hybridization buffer) were incubated at 40 °C overnight. Preamplifier, amplifier, and label probe oligonucleotides (diluted 1:25 in respective amplification buffers) were incubated each for 1 h at 40 °C. Following the washing steps, cells were immunostained for Tau to visualize the neurite boundaries.

Immunohistochemistry

For immunohistochemistry, TVA muscles were dissected from P5 Smn KO mice, and diaphragm tissues were dissected from P0 Munc13-1 KO and P5 Munc13-2 KO mice. Dissected muscle preparations were transferred into an extracellular physiological solution (135 mM NaCl, 12 mM NaHCO3, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 20 mM glucose) and fixed with 4% PFA at 4 °C for 90 min. PFA was quenched by 30 min incubation with 0.1 M glycine followed by permeabilization steps with 1% Triton X-100 (twice for 5 min, twice for 10 min, and twice for 30 min). Block solution (5% BSA, 0.1% Triton X-100 in PBS) was added and incubated at RT for 3 h. Primary antibodies diluted in block solution were incubated for two nights at 4 °C. Following wash steps with 0.1% Triton X-100 in PBS at RT, secondary antibodies were added together with α-Bungarotoxin (ThermoFisher Scientific, B13422, 1:1000) and incubated at RT for 1 h. Preparations were washed with 0.1% Triton X-100 in PBS, rinsed briefly in water, and embedded with Aqua-Poly/Mount. Alexa Fluor 488-conjugated α-Bungarotoxin was used to label postsynaptic membranes (AChRs) in NMJs. All the incubation steps were performed on a shaker. Following primary and secondary antibodies were used: guinea pig polyclonal anti-Synaptophysin1 (Synaptic Systems, 101004, 1:1000), rabbit polyclonal anti-Ca2+ channel P/Q-type (Cav2.1) (Synaptic Systems, 152203, 1:500), rabbit polyclonal anti-Munc13-1 (Synaptic System, 126103, 1:400), rabbit polyclonal anti-Munc13-2 (Synaptic System, 126203, 1:400), guinea pig polyclonal antiserum anti-Munc13-1 (Synaptic Systems, 126104, 1:500), donkey anti-rabbit IgG (H + L) AffiniPure (Cy3, Jackson ImmunoResearch, 711-165-152, 1:500), donkey anti-guinea pig IgG (H + L) AffiniPure (Cy5, Jackson ImmunoResearch, 706-175-148, 1:500).

Ca2+ imaging and data quantification

For Ca2+ imaging with cultured motoneurons, calcium indicator Oregon Green™ 488 BAPTA-1, AM, cell-permeant (ThermoFisher Scientific, O6807) was used. The calcium indicator was dissolved in Pluronic F-127/DMSO and sonicated in an ultrasonic bath for 2 min to prepare a 5 mM stock solution. Following twice washing steps with pre-warmed Ca2+ imaging buffer (135 mM NaCl, 6 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, and 5.5 mM glucose), Ca2+ indicator (5 µM diluted in Ca2+ imaging buffer) was added into cultured motoneuron dishes and incubated with for 15 min at 37 °C in a CO2 incubator. Residual calcium indicator dye was removed by twice washing with Ca2+ imaging buffer and cells were imaged in 2 ml Ca2+ imaging buffer supplemented with 3.5 ng/ml BDNF. A Nikon inverted epifluorescence microscope (TE2000) was used for time-lapse imaging. This was equipped with a 60 × 1.4-NA objective, a perfect focus system, an Orca Flash 4.0 V2 camera (Hamamatsu Photonics), an LED fluorescence light for excitation at 470 nm, and Nikon Element image software. Cells were imaged at 37 °C with 5% CO2 using a TOKAI HIT CO, LTD heated stage chamber. To monitor the spontaneous Ca2+ spikes, time-lapse images were taken at 500 ms intervals for 7 min. For membrane depolarization with KCl, cells were first imaged at 500 ms intervals for 1 min and then received 10 µl of 90 mM KCl, followed by another minute of imaging. Images were taken at 16-bit with a resolution of 1.024 × 1.024-pixel and a 2 × 2 binning. Quantifications of Ca2+ spikes were conducted in regions of interest (ROIs) within growth cones using Fiji. For this, intensity values were first measured from all time-lapse frames using the Fiji plug-in “dynamic Z-axis profile”. The measured average intensities were normalized to the average intensities of the first 10 frames before a spontaneous Ca2+ spike appeared (F0) and plotted (F/F0). For KCl pulse experiments, the measured time-lapse intensities were normalized to the average of the first 20 frames immediately before KCl application (F0) and plotted (F/F0). BAR Plugin of Fiji was used for counting the Ca2+ spikes.

RNA extraction and quantitative RT-PCR (qRT-PCR)

For RNA extraction from total cell lysates, spinal cord, and brain tissues, NucleoSpin RNA purification kit (MACHEREY-NAGEL, 740955.50) was used. RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, K1621) was used for reverse transcription with random primers. RNA extraction from microfluidic chambers was applied as previously described [8]. qRT-PCR was performed on a LightCycler 1.5 thermal cycler (Roche) using Luminaris HiGreen qPCR Master Mix (ThermoFisher Scientific, K0992). The relative expression was measured according to the ΔΔCt method using Gapdh for data normalization.

Following primers were used for qRT-PCR: Gapdh (forward) 5’-AACTCCCACTCTTCCACCTTC-3’ and (reverse) 5’-GGTCCAGGGTTTCTTACTCCTT-3’, Munc13-1 (forward) 5’-CACCACGCCCACCTACTGCTA-3’ and (reverse) 5’-TTGCGCTCGCGGATCT-3′, Munc13-2 (forward) 5’-CTTGGCAGATGATAATGAGTA-3’ and (reverse) 5’-GGTAGTCACTGTCTCGGTC-3′, Liprinα1 (forward) 5’-GATGGACTGCTTGACGGAAAC-3’ and (reverse) 5’-GGCCATTGCTTCACGGAC-3′, Bsn (forward) 5’-GCTGCCAGCCAACCAG-3’ and (reverse) 5’-CCACCAGGGAGGATCTTAGAG-3’, Pclo (forward) 5’-CCCGACCCATCCAAGGATATG-3’ and (reverse) 5’-TGGTTGAATGCGGAGTTGCT-3’, and RIM2 (forward) 5’-CAGACCCTGGCTACTCCTGC-3’ and (reverse) 5’-TACGGTGCTGGCAGTGTCTTG.

Western blotting

For Western blotting of primary mouse motoneurons, 300,000 cells were plated and grown for 7 days. Cells were lysed directly in 1 × Laemmli buffer (125 mM Tris, pH 6.8, 10% SDS, 50% glycerol, 25% β-mercaptoethanol, and 0.2% bromophenol blue). Lysates were boiled at 99 °C for 5 min, briefly centrifuged, and loaded onto 4–12% gradient SDS-PAGE gels. PVDF membranes were applied for blotting. For Western blot with brain and spinal cord, tissues were lysed in RIPA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% Sodium Deoxycholate, 0.1% SDS, 140 mM NaCl), protein concentration was measured using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, A55860), and 20 µg total protein was loaded. For Western blot with crude synaptosome fractions, cortices were dissected from P0 Munc13-1 KO and P5 Munc13-2 KO mice and synaptosome fractions were prepared as previously described (55). Briefly, cortices were homogenized in 500 µl cold sucrose lysis buffer (0.32 M sucrose, 5 mM HEPES, 1 × protease inhibitor cocktail (Roche)) and centrifuged at 1000 × g for 10 min at 4 °C. Pellets (P1) were discarded and supernatants (S1) were centrifuged at 12,000 × g for 20 min at 4 °C. Resulting supernatants (S2) were discarded and pellets (P2) containing crude synaptosomes were resuspended in 100 µl PBS. Protein concentration was measured and 20 µg total protein was loaded onto 4–12% SDS gels. Primary antibodies were incubated overnight at 4 °C, and secondary antibodies were incubated for 1 h at RT. ECL reagents (GE Healthcare) were used for the membrane developing. The following antibodies were used: rabbit polyclonal anti-Calnexin (Enzo Life Sciences, ADI-SPA-860-F, 1:6000), rabbit polyclonal anti-Munc13-1 (Synaptic Systems, 126103, 1:5000), rabbit polyclonal anti-Munc13-2 (Synaptic Systems, 126203, 1:5000), mouse monoclonal anti-α-Tubulin (Sigma-Aldrich, T5168, 1:5000), mouse monoclonal anti-β-actin (GeneTex, GTX26276, 1:5000), mouse monoclonal anti-SMN (BD Biosciences, 610647, 1:5000), guinea pig polyclonal anti-Synaptophysin1 (Synaptic Systems, 101004, 1:5000), peroxidase AffiniPure donkey anti-goat IgG (H + L) (Biozol, 705-035-003, 1:10000), peroxidase AffiniPure donkey anti-mouse IgG (H + L) (Biozol, 715-035-151, 1:10000), peroxidase AffiniPure goat anti-guinea pig IgG (H + L) (Jackson, 106-035-003, 1:10000), and peroxidase AffiniPure goat anti-rabbit IgG (H + L) (Biozol, 111-035-144, 1:10000).

Plasmid cloning of shRNA constructs targeting Munc13s and lentivirus production

For cloning of Munc13-1 overexpression lentivirus construct, a plasmid harboring the coding region (cDNA) of endogenous mouse Munc13-1 was purchased from GenScript and inserted into a lentivirus backbone vector with the Ubiquitin promotor using NEBuilder® HiFi DNA Assembly Cloning Kit (New England Biolabs, E5520S). The pSIH-H1 vectors with shRNA-targeting mouse Munc13-1 and Munc13-2 were generated as previously described [8]. The sequences of the antisense oligos used for shRNA cloning are as follows; Munc13-1: 5’-TCCCGTGTGAAACAAAGGT-3’, and Munc13-2: 5’-CGGAATAAACCAGAGATCT-3’. Lentiviruses were produced in HEK293T cells using TransIT-293 (Mirus, MIR2706) for transfection [25, 26]. pCMV-VSVG and pCMVΔR8.91 were employed as helper plasmids for the lentivirus production, and viral supernatants were collected by ultracentrifugation 60–72 h after transfection. The virus titer was assessed in NSC34 cells using serial dilutions.

Image acquisition and processing

Images were acquired with an Olympus Fluoview 1000 confocal microscope equipped with a 60 × 1.35-NA oil objective. For cultured motoneurons, 16-bit images with a resolution of 800 × 800 pixels were taken from single z-stacks. For neuromuscular junctions (NMJs), confocal imaging involved 6 z-stacks at 0.5 μm intervals, with maximum projection images presented for illustration. Super-resolution SIM imaging was performed with an ELYRA S.1 SIM, using a Plan-Apochromat 63× NA 1.4 oil objective and excitation lasers with wavelengths of 405 nm (50 mW), 488 nm (100 mW), 561 nm (100 mW), and 642 nm (150 mW). Laser power was adjusted between 2 and 5% with an integration time of 200 ms. For SIM, z-stacks of 110 nm intervals were captured, and maximum projection images were used for representation. The 16-bit raw images were processed with Zeiss ZEN 3.0 SR FP2 black software to reconstruct super-resolution images. Channel alignment was performed using fiducial markers (ThermoFisher Scientific, TetraSpeck™ Microspheres, 0.2 μm, fluorescent blue/green/orange/dark red, T7280). Fiji was used for image processing and analysis. Linear contrast enhancement was implemented to all representative images using Adobe Photoshop 24.2.0 for improved visibility.

Data analysis

To quantify immunofluorescence signals in growth cones and somata, mean gray values from unprocessed raw images were measured using Fiji after background subtraction. For assessing immunofluorescence signals at NMJs, average projections from multiple z-stack images were first created, and mean gray values were measured within the SynPhy-positive regions. All intensity measurements were normalized to the average intensity of the control group from the same experiment. For NMJ colocalization analysis, average z-stack projections were generated using Fiji, with NMJs defined as the region of interest, and Pearson R-values were calculated using Fiji. For the colocalization analysis of SIM data, single optical sections of 16-bit raw images were used to compute the Pearson R-value with Fiji, defining the entire growth cone as the region of interest. All immunostaining experiments were performed and analyzed in a blinded manner.

Statistical analysis

For creating graphs and performing statistical analyses GraphPad Prism 10 was utilized. Data are presented as bar graphs or scatter dot plots, with error bars indicating mean ± SEM, or as violin plots, with the median shown as dashed lines. Individual data points are shown as symbols, always when n ≤ 10. Statistical significance between two groups was assessed using the Mann-Whitney U test. For all experiments, the “n” number refers to the total number of analyzed cells, and the “N” number refers to the number of independent biological replicates. In most experiments, data were obtained from cultures prepared from at least N = 3 independent mice, with each mouse contributing one culture. Therefore, in most experiments, the total number of cultures used for analysis was N = 3, derived from N = 3 independent biological samples. Statistical analyses were performed based on these independent biological replicates rather than individual observations or technical replicates within the same experiment. For Ca2+ imaging experiments, statistical analysis were performed on pooled data from all four independent experiments.