Loss of Synaptic Munc13-1 Underlies Neurotransmission Abnormalities in Spinal Muscular Atrophy

Loss of Synaptic Munc13-1 Underlies Neurotransmission Abnormalities in Spinal Muscular Atrophy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Loss of Synaptic Munc13-1 Underlies Neurotransmission Abnormalities in Spinal Muscular Atrophy Mehri Moradi, Chunchu Deng, Michael Sendtner This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5305306/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Spinal muscular atrophy (SMA) is a devastating neurodegenerative disease characterized by degeneration of spinal motoneurons, leading to muscle atrophy and synaptic loss. SMN functions in mRNA splicing, transport, and local translation are crucial for maintaining synaptic integrity. Within the presynaptic membrane, the active zone orchestrates the docking and priming of synaptic vesicles. The Munc13 family proteins are key active zone components that operate precise neurotransmitter release in conjunction with voltage-gated Ca 2+ channels (VGCCs). However, the role of Munc13s in synaptic dysfunction in SMA remains elusive. Our findings reveal that Munc13-1 loss, but not Munc13-2, is closely linked to synaptic aberrations in SMA. Specifically, Munc13-1 mRNA localization in axons is dependent on Smn, and its disruption leads to impaired AZ assembly and VGCC clustering in motoneurons, ultimately reducing neuronal excitability. In contrast, Munc13-2 does not appear to be essential for AZ assembly or motoneuron differentiation, as its functions can be compensated by Munc13-1. These findings highlight the pivotal role of Munc13-1 in synapse integrity and point to potential therapeutic targets for mitigating synaptic loss in SMA. Active Zone Assembly Axonal mRNA Transport Munc13s Spinal Muscular atrophy Synapse Degeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Impaired synaptic function and degeneration are common pathological features of neurodegenerative diseases, including Spinal muscular atrophy (SMA) [ 1 , 2 ]. SMA is the second most common fatal autosomal recessive genetic disease with an incidence of 1 per 6,000 births [ 3 ]. SMA is caused by deletions of the Survival Motor Neuron 1 ( SMN1 ) gene [ 3 ]. These mutations lead to degeneration of spinal motoneurons, loss of axons, denervation of neuromuscular junctions (NMJs), presynaptic accumulation of neurofilaments, endplate abnormalities, and muscle atrophy in SMA patients and animal models [ 4 – 6 ]. The SMN protein is required for the assembly of small nuclear ribonucleoproteins involved in pre-mRNA splicing [ 7 ] as well as regulation of axonal mRNA transport and local translation [ 8 , 9 ]. Mouse models of SMA exhibit atrophic and smaller synapses, which are associated with impaired neurotransmitter release [ 10 ], disturbed clustering of voltage-gated Ca 2+ channels (VGCCs) [ 11 ], and reduced evoked postsynaptic potential at neuromuscular endplates [ 12 ]. Motoneurons in SMA are believed to undergo degeneration in a "dying-back" manner, where the presynaptic terminal withdraws from the postsynaptic endplate, resulting in partially innervated endplates [ 13 ]. Additionally, the loss of axons in the ventral roots is observed to be more pronounced than the loss of motoneuron cell bodies, indicating a distal-to-proximal pattern of degeneration [ 13 ]. In SMA mice, NMJ defects manifest before the onset of clinical symptoms, suggesting that the NMJ represents an "early pathological target" in SMA [ 14 ]. Despite the emerging evidence about synaptic impairments in SMA, the cellular mechanisms underlying synapse dysfunction and degeneration are not well understood. Neurotransmitter release at the presynaptic membrane is a finely tuned process initiated by Ca 2+ entry through VGCCs [ 15 ]. This occurs at specialized regions within the presynaptic plasma membrane known as active zones (AZs) [ 16 ]. At the molecular level, AZs are densely populated with a complex array of proteins such as RIM, Munc13s, Piccolo (Pclo), and Bassoon (Bsn), which interact with synaptic vesicles (SVs) to orchestrate their docking, priming, and fusion with the plasma membrane [ 17 ]. At the AZ within the presynaptic membranes, the Munc13 family release factor proteins achieve the temporal and spatial precision of SV release events in coordination with VGCCs [ 15 , 16 ]. Munc13-1 plays a crucial role in synaptic plasticity by regulating SV priming and modifying the fusion competence of the readily releasable pool [ 10 , 18 ]. Munc13-1 KO mice exhibit severe neurological phenotypes including a complete failure of neurotransmitter release at central synapses [ 17 ] and NMJs [ 18 ], leading to paralysis and early postnatal death [ 19 ]. In humans, UNC13A mutations are associated with microcephaly, cortical hyperexcitability, and fatal myasthenia [ 20 ]. Thus, we asked whether alterations in Munc13s synaptic functions might be associated with pathomechanisms underlying neurotransmission and plasticity defects in SMA. Here, we elucidated the roles of Munc13-1 and Munc13-2 in neurotransmission in a mouse model of SMA and revealed that loss of Munc13-1, but not Munc13-2, is associated with synaptic aberrations in SMA mice. Our findings indicate that axonal localization of Munc13-1 and Munc13-2 mRNAs depends on Smn and is perturbed in SMA. Additionally, we show that loss of Munc13-1 leads to defective organization of the AZ and VGCCs in presynaptic membranes in motoneurons, contributing to impaired excitability. Together, these data demonstrate a role for Munc13-1 in AZ assembly and neurotransmitter release in motoneurons, highlighting a potential mechanism for synaptic abnormalities in SMA. Materials and Methods 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/6N/Smn1tm1Hung Tg(SMN2)2Hung/J line with C57BL/6J background [ 21 ]. SMA litters ( Smn −/− , Hung tg/+ , or Smn KO), and control litters ( Smn +/− , Hung tg/+ or control) were derived from cross-breeding of Smn +/− to Smn −/− , Hung tg/tg . C57BL/6J mice were used as WT for control experiments. Munc13-1 KO mice ( Munc13-1 −/− ) , and Munc13-2 KO mice (Munc13-2 −/− ) [ 17 , 19 ] were obtained from Goettingen, Germany and cross-bred in-house. Enrichment and culturing of primary mouse motoneurons Primary mouse motoneurons were enriched via p75 NTR antibody panning and cultured as previously described [ 9 , 22 , 23 ]. For lentivirus transduction, cell suspensions were briefly exposed to lentiviral particles for 10 minutes 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 [ 11 , 24 ]. Cells were grown onto glass coverslips for immunofluorescence, 24-well plates for Western blot and qRT-PCR, and µ-dishes (Ibidi, 81156) for Ca 2+ imaging. Motoneuron culturing into compartmentalized microfluidic chambers was conducted as described earlier [ 25 ]. 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-Ca 2+ channel N-type alpha-1B (Ca v 2.2) (Synaptic System, 152305, 1:250), 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 [ 9 ]. 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 MgCl 2 , 2 mM CaCl 2 , 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-Ca 2+ channel P/Q-type (Ca v 2.1) (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). Ca imaging and data quantification For Ca 2+ 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 Ca 2+ imaging buffer (135 mM NaCl, 6 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 10 mM HEPES, and 5.5 mM glucose), Ca 2+ indicator (5 µM diluted in Ca 2+ imaging buffer) was added into cultured motoneuron dishes and incubated with for 15 min at 37°C in a CO 2 incubator. Residual calcium indicator dye was removed by twice washing with Ca 2+ imaging buffer and cells were imaged in 2 ml Ca 2+ 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% CO 2 using a TOKAI HIT CO, LTD heated stage chamber. To monitor the spontaneous Ca 2+ 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 Ca 2+ 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 Ca 2+ spike appeared (F 0 ) and plotted (F/F 0 ). For KCl pulse experiments, the measured time-lapse intensities were normalized to the average of the first 20 frames immediately before KCl application (F 0 ) and plotted (F/F 0 ). BAR Plugin of Fiji was used for counting the Ca 2+ 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 [ 9 ]. 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 12000 × 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), 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 [ 9 ]. 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 HEK 293T cells using TransIT-293 (Mirus, MIR2706) for transfection [ 26 , 27 ]. 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 NSC 34 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. 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. Statistical significance between two groups was assessed using the Mann-Whitney U test, while comparisons among multiple groups involved either the Mann-Whitney U test or one-way ANOVA followed by the Kruskal-Wallis test with Dunn’s Multiple Comparison post-hoc test. Results Transcripts for Munc13s, and Liprinα1, but not Pclo, Bsn, and RIM are enriched in motor axons RNA sequencing studies have shown that transcriptomes of neuronal processes, dendrites [ 28 , 29 ], and axons [ 25 , 30 , 31 ] include transcripts encoding synapse-related proteins. The Smn protein plays a key role in axonal mRNA transport and local translation of various targets [ 8 , 9 ], and the disruption of these functions is linked to impaired synaptic transmission in SMA [ 11 , 32 ]. This raises the possibility that improper mRNA localization of AZ components may contribute to synapse dysfunction in SMA. To explore this hypothesis, we investigated the localization of transcripts for AZ proteins within cultured mouse motoneurons using compartmentalized microfluidic chambers (Fig. 1 A). Quantitative real-time PCR (qRT-PCR) analysis revealed a significant enrichment of transcripts for the AZ proteins including Munc13-1, and Liprinα1 in distal axons compared to the somatodendritic compartment (Fig. 1 B). Although Munc13-2 transcripts also showed enrichment in axons, this was not statistically significant (Fig. 1 B). In contrast, transcripts for Pclo were significantly enriched in the somatodendritic compartment (Fig. 1 B). To further validate these findings, we employed single-molecule fluorescence in situ hybridization (smFISH) to visualize the subcellular localization of these transcripts. The smFISH results confirmed the localization of Munc13-1, Munc13-2, and Liprinα1 transcripts in distal axons in cultured motoneurons (Fig. 1 C). The assembly of the presynaptic active zone is diminished in Smn KO motoneurons Next, we assessed the impact of Smn loss of function on the axonal localization of transcripts for AZ proteins in cultured spinal motoneurons. To this end we employed a previously described and validated shRNA lentiviral construct to specifically target the mouse Smn [ 25 ]. Following Smn knockdown, qRT-PCR analysis demonstrated a significant reduction in the levels of Munc13-1, Munc13-2, and Liprinα1 transcripts in axons of cultured motoneurons (Fig. 1 D). These data suggest that Smn is crucial for the proper mRNA localization of these AZ components in motor axons. Since Smn is also involved in mRNA splicing, we investigated whether the loss of Smn affects the overall expression of AZ proteins. qRT-PCR analysis revealed a slight but statistically significant reduction in the total expression of AZ components in the brains of Smn KO mice, while no significant changes were observed in the spinal cord (Fig. S1A). These slight alterations, however, could be attributed to the severe synaptic loss observed in the late stages of SMA using P10 Smn KO mice. To evaluate this possibility, we analyzed the total expression of Munc13 proteins in cultured motoneurons from E13 Smn KO mice using qRT-PCR. The results showed no significant reduction in Munc13-1 and Munc13-2 mRNA levels in Smn KO motoneurons at embryonic stages (Fig. S1B). We then examined the distribution and levels of key AZ proteins in cultured motoneurons derived from Smn KO mice. Immunostaining revealed a marked reduction in Munc13-1 and Munc13-2 protein levels in Smn KO motoneurons (Fig. 2 A-D). Importantly, these reductions were observed specifically in axonal growth cones, with no significant changes detected in cell bodies (Fig. 2 B, D). To confirm these findings, we conducted Western blot assays on total lysates from cultured Smn KO motoneurons, as well as brain and spinal cord tissues from Smn KO mice. The results showed a minor reduction in Munc13-1 protein levels in cultured Smn KO motoneurons (Fig. S1C), but no significant changes in its levels in brain and spinal cord tissues (Fig. S1D, E). In contrast, Munc13-2 protein levels were elevated in total lysates from cultured Smn KO motoneurons (Fig. S1F), as well as in brain (Fig. S1G) and spinal cord tissues (Fig. S1H) of Smn KO mice, suggesting a compensatory upregulation mechanism. Further analysis of other AZ components including Pclo, RIM1/2, Bsn, and Liprinα1 revealed a broader disruption in AZ protein organization within the growth cones of Smn KO motoneurons (Fig. 2 E-J). Additionally, we assessed the levels of Munc13 isoforms at NMJs from the transverse abdominis muscles (TVA) of Smn KO mice. Notably, we observed a significant reduction in Munc13-1 levels at NMJs, while Munc13-2 levels remained unaltered (Fig. 3 A-D). The specificity of the Munc13-1 and Munc13-2 antibodies was validated by Western blot analysis of synaptosome fractions from the cortices of Munc13s KO mice (Fig. S2A, B), as well as by immunohistochemistry analysis of NMJs from Munc13s KO mice (Fig. S2C, D). These findings suggest that loss of Smn severely impairs the organization of AZ components within the presynaptic membranes, in particular Munc13-1. This disruption likely contributes to the synaptic abnormalities seen in SMA, underscoring the vulnerability of the synaptic assembly process in the absence of a functional Smn protein. Munc13-1 tethers VGCCs into presynaptic AZs in motoneurons At presynaptic AZs, Munc13-1 interacts with proteins like RIM and RIM binding protein (RIM-BP), forming a complex that helps tether SVs close to VGCCs [ 17 , 18 ]. This precise spatial alignment between Munc13s and VGCCs ensures temporal and spatial regulation of neurotransmitter release [ 19 , 20 ]. To gain a detailed view of the spatial colocalization between Munc13 isoforms and Ca v 2.2 (a subtype of VGCCs) in axonal growth cones of wildtype (WT) motoneurons, we employed Structured Illumination Microscopy (SIM) (Fig. 3 E). Quantitative analysis revealed that VGCCs (Ca v 2.2) colocalize significantly more with Munc13-1 than Munc13-2 (Fig. 3 F). This finding is consistent with previous studies that demonstrated a tighter coupling between Unc13a and VGCCs at AZs in Drosophila NMJs [ 15 ]. Intriguingly, SIM imaging showed reduced colocalization between Munc13-1 and Ca v 2.2 at AZs in cultured Smn KO motoneurons (Fig. 3 G, H). This reduction indicates a disruption in the spatial organization of these critical synaptic components. Similarly, confocal imaging of NMJs from the TVA muscles of Smn KO and control mice showed a significant decrease in the colocalization between Munc13-1 and Ca v 2.1 (another VGCC subtype) at NMJs in Smn KO mice (Fig. 3 I, J). These findings suggest that the loss of Smn disrupts the spatial organization of Munc13-1 and VGCCs at both axonal growth cones and NMJs, potentially contributing to the synaptic dysfunction in SMA. Depletion of Munc13-1 affects the AZ assembly and diminishes neuronal excitability We investigated the role of Munc13-1 and Munc13-2 in motoneuron development and function by employing shRNA constructs to selectively knock down these proteins. Additionally, we analyzed the phenotypes of cultured motoneurons obtained from Munc13s KO mice. As shown in Fig. 4 A, Western blot analysis confirmed effective knockdown of Munc13-1 in motoneurons transduced with shRNA targeting Munc13-1. Imaging of axonal growth cones revealed that Munc13-1-depleted motoneurons exhibited significantly smaller growth cones compared to controls (Fig. 4 B, C), suggesting impaired growth cone differentiation. Despite this reduction in growth cone size, axon outgrowth was unaffected by Munc13-1 depletion (Fig. 4 D). Interestingly, measurements of growth cone area and axon length in Munc13-1 KO motoneurons were comparable to those in control motoneurons (Fig. 4 E-G). This discrepancy may be due to compensatory mechanisms activated in Munc13-1 KO motoneurons. Furthermore, to elucidate whether Munc13-1 loss recapitulates SMA phenotype, we examined the AZ components and VGCCs in Munc13-1 KO motoneurons by immunostaining (Fig. 4 H, K). These quantitative analyses showed that Ca v 2.2 levels were reduced in axonal growth cones of Munc13-1 KO motoneurons (Fig. 4 H, I), while Munc13-2 levels remained unchanged (Fig. 4 H, J). In line with this, the levels of RIM1/2, Pclo, and Bsn were reduced in axonal growth cones of Munc13-1 KO motoneurons (Fig. 4 K-N). Intriguingly, these abnormalities in AZ assembly and VGCC clustering correlated with impaired excitability in Munc13-1 KO motoneurons. As demonstrated by Ca 2+ imaging, Munc13-1 KO motoneurons exhibit reduced spontaneous Ca 2+ transients in growth cones (Fig. 5 A, B), although the amplitude of these transients was not altered (Fig. 5 C). Additionally, we applied a KCl depolarization to measure the evoked Ca 2+ transient response (Fig. 5 D). The maximum response to induced depolarization was diminished in growth cones of Munc13-1 KO motoneurons (Fig. 5 E, F), and the percentage of failed responses to membrane depolarization was higher (Fig. 5 G). In summary, these findings indicate that depletion of Munc13-1 leads to defects in growth cone differentiation, impaired AZ assembly, and abnormal VGCC clustering, which together contribute to reduced neuronal excitability in motoneurons. Munc13-2 synaptic functions are compensated by Munc13-1 To assess the role of Munc13-2 in motoneurons, we knocked down Munc13-2 using lentivirus transduction and analyzed its subsequent effects on AZ assembly and axonal growth and differentiation. Western blot analysis confirmed a reduction in Munc13-2 protein levels in motoneurons transduced with shRNA targeting Munc13-2 (Fig. 6 A). Downregulation of Munc13-2 did not affect the area of axonal growth cones but impaired axon outgrowth in cultured motoneurons (Fig. 6 B-D). Next, we analyzed the axon growth and differentiation in cultured Munc13-2 KO motoneurons. Our data revealed that the area of axonal growth cones and the axon length of Munc13-2 KO motoneurons were comparable to those of the control (Fig. 6 E-G). Moreover, immunostaining of AZ proteins revealed unaltered levels of AZ components in axonal growth cones of cultured Munc13-2 KO motoneurons (Fig. 6 H-N), suggesting that Munc13-2 is not essential for AZ assembly or growth cone differentiation in spinal motoneurons. To explore potential compensatory mechanisms for Munc13-2 loss, we analyzed the effects of Munc13-1 overexpression in motoneurons. For this purpose, we generated a lentiviral construct expressing Munc13-1 (Munc13-1 OE ) and confirmed its overexpression in cultured motoneurons through immunostaining and Western blot assays (Fig. S3A-C). Immunostaining revealed that upon transduction of motoneurons with Munc13-1 OE virus, Munc13-2 levels decreased in axonal growth cones, while its levels remained unaltered in the soma (Fig. 7 A-C). To further verify that Munc13-1 overexpression does not alter the total expression of Munc13-2, we conducted qRT-PCR and Western blot. The obtained data revealed no significant changes in Munc13-2 mRNA levels in total lysates from Munc13-1 OE -transduced motoneurons (Fig. 7 D). This indicates that the specific reduction in Munc13-2 protein in axonal growth cones was not due to its transcriptional downregulation. Western blot analysis further confirmed that overall protein levels of Munc13-2 remain unaltered by Munc13-1 overexpression (Fig. 7 E, F). Interestingly, unlike Munc13-2, overexpression of Munc13-1 in motoneurons led to increased levels of Ca v 2.2 in axonal growth cones (Fig. S3D) without affecting the overall levels of other AZ proteins (Fig. S3E-H). These data unravel a specific and non-redundant role for Munc13-1 in VGCC tethering. To determine whether a similar compensatory mechanism occurs for Munc13-1 loss through Munc13-2, we measured the Munc13-2 levels in cultured Munc13-1 KO motoneurons. Notably, we observed that Munc13-2 levels remained unchanged in axonal growth cones of Munc13-1 KO motoneurons (Fig. 4 H, J). Consistent with this finding, total Munc13-2 levels were not elevated in Munc13-1 KO motoneurons, as demonstrated by immunostaining of cell bodies and Western blot analysis (Fig. 7 G-J). In conclusion, while Munc13-2 downregulation affects axon growth, it does not disrupt AZ assembly or growth cone differentiation. Furthermore, these data suggest that Munc13-1 has redundant functions with Munc13-2 and can compensate for Munc13-2 in motoneurons, thereby maintaining synaptic functions despite the loss of Munc13-2. Discussion The present study provides new insights into the role of Munc13 proteins in the pathogenesis of synaptic dysfunctions in SMA. Our findings reveal that Munc13-1, but not Munc13-2, plays a critical role in the organization of AZs and the clustering of VGCCs in motoneurons, suggesting that Munc13-1 dysregulation contributes to the synaptic defects observed in SMA. A common therapeutic strategy for SMA involves increasing SMN protein levels through gene therapy approaches, referred to as SMN-based therapies. These treatments target the SMN2 gene to enhance SMN protein production [ 33 ]. However, non-responsive cases often arise due to two main factors: patients who miss the therapeutic window benefit less from SMN-repletion therapies, and there is still a significant knowledge gap in understanding how SMN deficiency leads to NMJ dysfunction and synapse degeneration. Understanding the cellular mechanisms driving synaptic deficiencies is therefore critical for identifying disease modifiers and developing combination therapies to improve patient outcomes. Our findings indicate that Munc13-1 depletion in motoneurons results in aberrant assembly of presynaptic AZs and impairs clustering of VGCCs in axonal growth cones, leading to reduced neuronal excitability. This is consistent with previous studies highlighting the Munc13-1 ability to tether VGCCs to SVs [ 15 , 34 ], and defective neurotransmitter release in its absence in hippocampal neurons and at neuromuscular junctions [ 17 , 19 ]. In line with this, we observed reduced Munc13-1 levels in presynaptic terminals of cultured Smn KO motoneurons and at NMJs in Smn KO mice. These findings provide a molecular mechanism that links Smn deficiency to synaptic abnormalities in SMA. Interestingly, although Munc13-2 also localizes to axonal growth cones, its role appears to be less crucial in motoneurons. Our data show that the loss of Munc13-2 does not significantly affect AZ assembly or growth cone differentiation, suggesting that Munc13-1 can compensate for the absence of Munc13-2. Conversely, in Munc13-1 KO motoneurons, the Munc13-2 levels remained unchanged, suggesting that Munc13-1 cannot be fully compensated by Munc13-2 in motoneurons. This lack of compensation may contribute to the synaptic deficits observed in SMA, emphasizing the non-redundant function of Munc13-1 in maintaining synaptic function. Moreover, the failure to upregulate Munc13-2 in response to Munc13-1 loss suggests that therapeutic strategies aimed at increasing Munc13-2 expression may not be sufficient to restore synaptic function in SMA. In neurons, subcellular mRNA localization and intra-axonal local translation are conserved cellular mechanisms that regulate synaptogenesis, plasticity, and axon regeneration through temporal and spatial modulation of the local proteome [ 35 – 37 ]. Disruption in local translation impacts neuronal function and viability, leading to neurodegeneration in ALS, SMA, Fragile X Syndrome, Alzheimer's disease, and Parkinson's disease [ 35 , 38 , 39 ]. Local translation is particularly crucial for spinal motoneurons due to their long axons and their unique anatomical and morphological characteristics [ 40 ]. These distinct features make motoneurons especially vulnerable to degeneration resulting from disruptions in protein synthesis, particularly within the axon. Impaired local translation in SMA is attributed to disrupted mRNA subcellular localization [ 30 , 41 ], and the loss of direct interaction between the SMN protein and ribosomes, which is essential for the recruitment of mRNA onto polysomes [ 42 ]. Moreover, loss of SMN is associated with disturbed ribosome assembly and impaired formation of the rough endoplasmic reticulum (RER) in presynaptic compartments in motoneurons, leading to delayed translation initiation [ 43 ]. Here, we demonstrate that Smn downregulation results in a significant reduction in Munc13-1 and Liprinα1 mRNAs in axons of primary cultured mouse motoneurons. This finding highlights the importance of mRNA transport and local translation of synaptic proteins for the maintenance of synaptic integrity. This aligns with previous reports that Smn plays a critical role in mRNA localization and that its deficiency leads to widespread defects in axonal protein synthesis [ 8 , 9 , 44 ]. The mechanisms by which Smn regulates the axonal translocation of Munc13-1 mRNA in motoneurons may involve the RNA-binding protein heterogeneous nuclear ribonucleoprotein R (hnRNP R). Importantly, iCLIP assays have revealed that Munc13-1 mRNA binds to hnRNP R via its 3' untranslated region (3'UTR) [ 45 ]. This interaction suggests that the correct axonal localization of Munc13-1 depends on the presence of hnRNP R within axons. hnRNP R is a binding partner of Smn, and in Smn KO motoneurons, axonal levels of hnRNP R are reduced [ 46 ]. Consequently, reduced levels of hnRNP R in axons of Smn KO motoneurons may disrupt the localization of Munc13-1 mRNAs within axons, potentially affecting axonal function. Thus, to fully restore synaptic function in SMA, therapies targeting SMN function may need to focus as well on correcting the mRNA transport and local translation processes. In conclusion, our findings demonstrate that Munc13-1 is pivotal for AZ assembly and neurotransmitter release in motoneurons, and its dysregulation contributes to the synaptic defects observed in SMA. The inability of Munc13-2 to fully compensate for Munc13-1 loss underscores the non-redundant functions of these proteins in maintaining synaptic integrity. These insights provide a deeper understanding of the molecular mechanisms underlying synaptic dysfunction in SMA and may inform the development of targeted therapies aimed at mitigating synaptic loss in this devastating disease. Declarations Acknowledgments We are grateful to Markus Sauer for his support with SIM, Michael Briese and Kilian Katzenberger for their technical assistance, Regine Sendtner for her work in animal breeding, and Hildegard Troll for her contribution to lentivirus production. Funding This project was funded by DFG, Grant Se697/7-1 to Michael Sendtner. Competing interests The authors declare no competing financial interests. Author contribution M. M. and M. S. conceived the project, designed the experiments, and prepared the manuscript. M. M. and CC. D. conducted the experiments. Data Availability All datasets are provided within the main text or Supplementary Materials and can be obtained from the corresponding authors upon request. This study includes no data deposited in external repositories. Ethics approval This study was performed in line with the principles of the Declaration of Germany. Approval was granted by the Ethics Committee of the University of Wuerzburg. References Bae, J.R. and S.H. Kim, Synapses in neurodegenerative diseases. BMB Rep, 2017. 50 (5): p. 237-246. Wilson, D.M., 3rd, et al., Hallmarks of neurodegenerative diseases. Cell, 2023. 186 (4): p. 693-714. Crawford, T.O. and C.A. Pardo, The neurobiology of childhood spinal muscular atrophy. Neurobiol Dis, 1996. 3 (2): p. 97-110. Goulet, B.B., R. Kothary, and R.J. Parks, At the "junction" of spinal muscular atrophy pathogenesis: the role of neuromuscular junction dysfunction in SMA disease progression. Curr Mol Med, 2013. 13 (7): p. 1160-74. Tisdale, S. and L. Pellizzoni, Disease mechanisms and therapeutic approaches in spinal muscular atrophy. J Neurosci, 2015. 35 (23): p. 8691-700. Wee, C.D., L. Kong, and C.J. Sumner, The genetics of spinal muscular atrophies. Curr Opin Neurol, 2010. 23 (5): p. 450-8. Pillai, R.S., et al., Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing. Genes Dev, 2003. 17 (18): p. 2321-33. Fallini, C., G.J. Bassell, and W. Rossoll, Spinal muscular atrophy: the role of SMN in axonal mRNA regulation. Brain Res, 2012. 1462 : p. 81-92. Moradi, M., et al., Differential roles of alpha-, beta-, and gamma-actin in axon growth and collateral branch formation in motoneurons. J Cell Biol, 2017. 216 (3): p. 793-814. Kong, L., et al., Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J Neurosci, 2009. 29 (3): p. 842-51. Jablonka, S., et al., Defective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy. J Cell Biol, 2007. 179 (1): p. 139-49. Xu, Y., et al., Defects in Neuromuscular Transmission May Underlie Motor Dysfunction in Spinal and Bulbar Muscular Atrophy. J Neurosci, 2016. 36 (18): p. 5094-106. Murray, L.M., et al., Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. Hum Mol Genet, 2008. 17 (7): p. 949-62. Courtney, N.L., et al., Reduced P53 levels ameliorate neuromuscular junction loss without affecting motor neuron pathology in a mouse model of spinal muscular atrophy. Cell Death Dis, 2019. 10 (7): p. 515. Bohme, M.A., et al., Active zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling. Nat Neurosci, 2016. 19 (10): p. 1311-20. Südhof, T.C., The presynaptic active zone. Neuron, 2012. 75 (1): p. 11-25. Augustin, I., et al., Munc13-1 is essential for fusion competence of glutamatergic synaptic vesicles. Nature, 1999. 400 (6743): p. 457-61. Varoqueaux, F., et al., Aberrant morphology and residual transmitter release at the Munc13-deficient mouse neuromuscular synapse. Mol Cell Biol, 2005. 25 (14): p. 5973-84. Varoqueaux, F., et al., Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming. Proc Natl Acad Sci U S A, 2002. 99 (13): p. 9037-42. Engel, A.G., et al., Loss of MUNC13-1 function causes microcephaly, cortical hyperexcitability, and fatal myasthenia. Neurol Genet, 2016. 2 (5): p. e105. Hsieh-Li, H.M., et al., A mouse model for spinal muscular atrophy. Nat Genet, 2000. 24 (1): p. 66-70. Deng, C., et al., Dynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons. J Cell Sci, 2021. 134 (22). Wiese, S., et al., Isolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos. Nat Protoc, 2010. 5 (1): p. 31-8. Chand, K.K., et al., Loss of β 2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction. J Physiol, 2015. 593 (1): p. 245-65. Saal, L., et al., Subcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation. RNA, 2014. 20 (11): p. 1789-802. Subramanian, N., et al., Role of Na(v)1.9 in activity-dependent axon growth in motoneurons. Hum Mol Genet, 2012. 21 (16): p. 3655-67. Rehberg, M., et al., A new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum. Cell Calcium, 2008. 44 (4): p. 386-99. Hobson, B.D., et al., Subcellular and regional localization of mRNA translation in midbrain dopamine neurons. Cell Rep, 2022. 38 (2): p. 110208. Cajigas, I.J., et al., The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron, 2012. 74 (3): p. 453-66. Hennlein, L., et al., Plastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy. J Cell Biol, 2023. 222 (3). Tushev, G., et al., Alternative 3' UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments. Neuron, 2018. 98 (3): p. 495-511.e6. Kong, L., et al., Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J Neurosci, 2009. 29 (3): p. 842-51. Jablonka, S. and M. Sendtner, Developmental regulation of SMN expression: pathophysiological implications and perspectives for therapy development in spinal muscular atrophy. Gene Ther, 2017. 24 (9): p. 506-513. Dittman, J.S., Unc13: a multifunctional synaptic marvel. Curr Opin Neurobiol, 2019. 57 : p. 17-25. Holt, C.E., K.C. Martin, and E.M. Schuman, Local translation in neurons: visualization and function. Nat Struct Mol Biol, 2019. 26 (7): p. 557-566. Kang, H. and E.M. Schuman, A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science, 1996. 273 (5280): p. 1402-6. Jung, H., et al., Remote control of gene function by local translation. Cell, 2014. 157 (1): p. 26-40. Lin, J.Q., F.W. van Tartwijk, and C.E. Holt, Axonal mRNA translation in neurological disorders. RNA Biol, 2021. 18 (7): p. 936-961. Shigeoka, T., et al., Dynamic Axonal Translation in Developing and Mature Visual Circuits. Cell, 2016. 166 (1): p. 181-92. Ragagnin, A.M.G., et al., Motor Neuron Susceptibility in ALS/FTD. Front Neurosci, 2019. 13 : p. 532. Briese, M., et al., Loss of Tdp-43 disrupts the axonal transcriptome of motoneurons accompanied by impaired axonal translation and mitochondria function. Acta Neuropathol Commun, 2020. 8 (1): p. 116. Lauria, F., et al., SMN-primed ribosomes modulate the translation of transcripts related to spinal muscular atrophy. Nat Cell Biol, 2020. 22 (10): p. 1239-1251. Deng, C., et al., Impaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus-response in spinal muscular atrophy. Transl Neurodegener, 2022. 11 (1): p. 31. Rossoll, W., et al., Smn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons. J Cell Biol, 2003. 163 (4): p. 801-12. Briese, M., et al., hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons. Proc Natl Acad Sci U S A, 2018. 115 (12): p. E2859-E2868. Rossoll, W., et al., Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum Mol Genet, 2002. 11 (1): p. 93-105. Supplementary Files SupplementaryFigures.docx Cite Share Download PDF Status: Published Journal Publication published 29 Aug, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 18 Dec, 2024 Reviewers agreed at journal 31 Oct, 2024 Reviewers invited by journal 30 Oct, 2024 Editor assigned by journal 28 Oct, 2024 First submitted to journal 21 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5305306","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":372396327,"identity":"ab2900fd-445a-45b4-b35c-ba7912d57aaf","order_by":0,"name":"Mehri Moradi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBAC+wYeAyDFzMMgkXyAsYGBAYTxAwMGvgKIFplnCcRq4f8A0sLAIP/GgFgtvBsfV9RYyzBI53yTnNnGINtPSIs9A+9mwzPH0nkYpHO3SW5sYzCeScgaoC3bJBvYDkO0PNzGkLjhAEEtPGaSDf+AWiRynoG17CdKS2MbWAub5EaQLYT8AtRibNjYB/SLRJqx5cx/EsYzCNkCjErDhw3frO2BUfnwZs8ZG9n+BkLWyD+A6oUYLkFI/SgYBaNgFIwCYgAA3hE88NK1pm0AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-9550-8701","institution":"Universitätsklinikum Würzburg: Universitatsklinikum Wurzburg","correspondingAuthor":true,"prefix":"","firstName":"Mehri","middleName":"","lastName":"Moradi","suffix":""},{"id":372396328,"identity":"e6c30023-5a93-4e6d-a1d5-2b555594ea9b","order_by":1,"name":"Chunchu Deng","email":"","orcid":"","institution":"Tongji Medical College of Huazhong University of Science and Technology: Huazhong University of Science and Technology Tongji Medical College","correspondingAuthor":false,"prefix":"","firstName":"Chunchu","middleName":"","lastName":"Deng","suffix":""},{"id":372396329,"identity":"ca1c2947-b019-42de-9618-fa2240d26599","order_by":2,"name":"Michael Sendtner","email":"","orcid":"https://orcid.org/0000-0002-4737-2974","institution":"Universitätsklinikum Würzburg: Universitatsklinikum Wurzburg","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Sendtner","suffix":""}],"badges":[],"createdAt":"2024-10-21 14:32:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5305306/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5305306/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05859-7","type":"published","date":"2025-08-29T15:58:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":68729955,"identity":"65ded19f-98cf-4e85-9658-1774e28e7347","added_by":"auto","created_at":"2024-11-11 12:12:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":301356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscripts for active zone proteins are enriched in distal axons of cultured mouse motoneurons.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Schematic representation of implied microfluidic chambers. \u003cstrong\u003eB\u003c/strong\u003e qRT-PCR indicates a significant relative enrichment of transcripts of Munc13-1 (**P = 0.004), and Liprinα1 (*P = 0.0476), and enrichment of transcripts of Munc13-2 in axons of cultured motoneurons. In contrast, transcripts of Pclo (**P = 0.004) are enriched in the somatodendritic compartment of cultured motoneurons (n = 3-5 cultures/mice). \u003cstrong\u003eC\u003c/strong\u003eRepresentative images of smFISH with cultured motoneurons showing axonal localization of transcripts of Munc13-1, Munc13-2, and Liprinα1, in cultured motoneurons. \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR shows reduced mRNA levels of Munc13-1 (**P = 0.0022, n = 5 cultures/mice), Munc13-2 (**P = 0.0016, n = 8 cultures/mice), and Liprinα1 (***P = 0.0006, n = 6 cultures/mice) in axons after Smn knockdown. In \u003cstrong\u003eB\u003c/strong\u003eand \u003cstrong\u003eD\u003c/strong\u003e, data are presented as mean ± SEM. *P ≤ 0.05 (One-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/49aa02e875bef75521d6a0fb.png"},{"id":68729948,"identity":"c7986ba5-4858-43a3-bbc6-0e6d72ad7f75","added_by":"auto","created_at":"2024-11-11 12:12:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":428202,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe active zone organization is impaired in cultured Smn KO motoneurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e,\u003cstrong\u003e C\u003c/strong\u003e,\u003cstrong\u003e E\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003eand\u003cstrong\u003e F\u003c/strong\u003e Representative images of axonal growth cones of cultured motoneurons from Smn\u003csup\u003e+/-\u003c/sup\u003e,Hung\u003csup\u003etg/+ \u003c/sup\u003eand\u003csup\u003e \u003c/sup\u003eSmn\u003csup\u003e-/-\u003c/sup\u003e,Hung\u003csup\u003etg/+ \u003c/sup\u003emice immunostained against active zone proteins. \u003cstrong\u003eB \u003c/strong\u003eand\u003cstrong\u003e D\u003c/strong\u003e Graphs show reduced Munc13-1 (\u003cstrong\u003eB\u003c/strong\u003e: ****P \u0026lt; 0.0001, n = 144-162 cells, n = 4 cultures/mice for each genotype), and Munc13-2 (\u003cstrong\u003eD\u003c/strong\u003e: ***P = 0.0003, n = 106-110 cells, n = 4 cultures/mice for each genotype) protein levels in axonal growth cones but not cell bodies of Smn KO\u003csup\u003e \u003c/sup\u003emotoneurons.\u003cstrong\u003e G\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e Graphs indicate significant reduction in protein levels of Pclo (\u003cstrong\u003eG\u003c/strong\u003e: **P = 0.0051, n = 58-60 cells), RIM1/2 (\u003cstrong\u003eH\u003c/strong\u003e: ***P = 0.0001, n = 96-99 cells), Bsn (\u003cstrong\u003eI\u003c/strong\u003e: **P = 0.0046, n = 101-122 cells), and Liprinα1 (\u003cstrong\u003eJ\u003c/strong\u003e: ****P \u0026lt; 0.0001, n = 74-88 cells) in axonal growth cones of cultured Smn KO motoneurons (n = 3-4 cultures/mice for each genotype). In \u003cstrong\u003eB, D, \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e-\u003cstrong\u003eJ\u003c/strong\u003e, data are presented as violin plots with the median shown as dashed lines. *P ≤ 0.05 (Two-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/9ff20423968a0cb9616fcbb8.png"},{"id":68729954,"identity":"42a816e9-8baa-4506-b64f-4e36503b3c63","added_by":"auto","created_at":"2024-11-11 12:12:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":541208,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe spatial colocalization between Munc13-1 and VGCCs is altered at NMJs from Smn KO mice.\u003c/strong\u003e \u003cstrong\u003eA \u003c/strong\u003eand\u003cstrong\u003e C\u003c/strong\u003e Representative images of NMJs from TVA muscles isolated from P5 control and Smn KO mice. \u003cstrong\u003eB\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Munc13-1 (\u003cstrong\u003eB\u003c/strong\u003e: ****P \u0026lt; 0.0001, n = 79-95 NMJs, n = 3 mice for each group), but not Munc13-2 (\u003cstrong\u003eD\u003c/strong\u003e:\u003cstrong\u003e \u003c/strong\u003eP = 0.357, n = 80-94 NMJs, n = 3 mice for each group) levels are reduced at NMJs in Smn KO mice. \u003cstrong\u003eE\u003c/strong\u003e Representative SIM images of axonal growth cones of cultured motoneurons stained against Munc13s and Ca\u003csub\u003ev\u003c/sub\u003e2.2. Inset on the right side: Zoom-in indicates colocalization between Munc13 isoforms and Ca\u003csub\u003ev\u003c/sub\u003e2.2. \u003cstrong\u003eF\u003c/strong\u003e Quantification of data represented in \u003cstrong\u003eE \u003c/strong\u003ereveals greater overlap between Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.2 than that between Munc13-2 and Ca\u003csub\u003ev\u003c/sub\u003e2.2, in axonal growth cones of cultured WT motoneurons (**P = 0.0021, n = 44-49 cells, n = 3 cultures/mice).\u003cstrong\u003e G\u003c/strong\u003e Representative SIM images of axonal growth cones of control and Smn KO motoneurons indicating Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.2 colocalization. \u003cstrong\u003eH\u003c/strong\u003e Quantification of data represented in \u003cstrong\u003eG \u003c/strong\u003edemonstrates reduced colocalization between Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.2 at axonal growth cones in Smn KO motoneurons compared to control (*P = 0.042, n = 13 cells, n = 2 cultures/mice for each genotype). \u003cstrong\u003eI\u003c/strong\u003e Representative images of NMJs from TVA muscles from P5 control and Smn KO mice. \u003cstrong\u003eJ\u003c/strong\u003e Quantification of data represented in \u003cstrong\u003eI \u003c/strong\u003eshows reduced colocalization between Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.1 at NMJs in Smn KO mice (**P = 0.0076, n = 50-65 NMJs, n = 3 mice for each group). Data are presented as violin plots with the median shown as dashed lines. *P ≤ 0.05 (Two-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/a7f1af1b60d9982ed51f17ec.png"},{"id":68730192,"identity":"f7cb8ca9-8494-4c9c-821d-bd0846c3176a","added_by":"auto","created_at":"2024-11-11 12:20:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":548363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMunc13-1 loss of function impairs the AZ assembly and synapse differentiation in motoneurons. A\u003c/strong\u003e Western blot with total lysates from cultured motoneurons transduced with lentiviruses of shCtrl and shRNA targeting Munc13-1.\u003cstrong\u003e B\u003c/strong\u003e Representative images of axonal growth cones of cultured motoneurons transduced with shCtrl or shMunc13-1 lentiviruses, stained against β-actin. \u003cstrong\u003eC\u003c/strong\u003e Munc13-1-depleted motoneurons exhibit smaller axonal growth cones (**P = 0.0017, n = 43-45 cells, n = 3 cultures/mice). \u003cstrong\u003eD\u003c/strong\u003e shRNA-mediated Knockdown of Munc13-1 does not affect axon outgrowth (P = 0.0641, n = 205-215 cells, n = 3 cultures/mice).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e Representative images of axonal growth cones of control and Munc13-1 KO motoneurons stained against β-actin. \u003cstrong\u003eF \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e The area of axonal growth cones (n = 35-38 cells), as well as the axon length (n = 98-105 cells), are comparable between control and Munc13-1 KO motoneurons (n = 3 cultures/mice for each genotype). \u003cstrong\u003eH \u003c/strong\u003eand\u003cstrong\u003e K\u003c/strong\u003e Representative images of axonal growth cones of cultured control and Munc13-1 KO motoneurons stained against AZ proteins and Ca\u003csub\u003ev\u003c/sub\u003e2.2.\u003cstrong\u003e I \u003c/strong\u003eand \u003cstrong\u003eJ\u003c/strong\u003e The immunoreactivity of Ca\u003csub\u003ev\u003c/sub\u003e2.2 (**P = 0.0064, n = 50-52 cells), but not Munc13-2 (n = 52-54 cells, n = 3 cultures/mice for each genotype) is decreased in axonal growth cones of Munc13-1 KO motoneurons.\u003cstrong\u003e L\u003c/strong\u003e-\u003cstrong\u003eN\u003c/strong\u003e Graphs demonstrate reduced immunoreactivity of RIM1/2 (\u003cstrong\u003eL\u003c/strong\u003e: ****P \u0026lt; 0.0001, n = 64-66 cells), Pclo (\u003cstrong\u003eM\u003c/strong\u003e: **P = 0.0064, n = 60-65 cells), and Bsn (\u003cstrong\u003eN\u003c/strong\u003e: *P = 0.0423, n = 64-66 cells) in axonal growth cones of Munc13-1 KO motoneurons (n = 3 cultures/mice for each genotype). Data are presented as violin plots with the median shown as dashed lines. *P ≤ 0.05 (Two-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/fac49a580f839e7ad843836d.png"},{"id":68729947,"identity":"05a0d502-d3b4-4091-b664-8d69c4e5f4b1","added_by":"auto","created_at":"2024-11-11 12:12:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":113519,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeuronal excitability is attenuated in\u003c/strong\u003e \u003cstrong\u003eMunc13-1 KO motoneurons. A \u003c/strong\u003eand\u003cstrong\u003e B\u003c/strong\u003e Ca\u003csup\u003e+\u003c/sup\u003e imaging reveals decreased spontaneous Ca\u003csup\u003e+\u003c/sup\u003e transients in axonal growth cones of Munc13-1 KO motoneurons (*P = 0.0383, n = 22-25 cells, n = 4 cultures/mice for each genotype).\u003cstrong\u003e C\u003c/strong\u003e The amplitude of spontaneous Ca\u003csup\u003e+\u003c/sup\u003e transients is not altered in Munc13-1 KO motoneurons (n = 27-55 Ca\u003csup\u003e+\u003c/sup\u003e transients from 22-25 cells, n = 4 cultures/mice for each genotype). \u003cstrong\u003eD\u003c/strong\u003e-\u003cstrong\u003eF\u003c/strong\u003e The maximum response to a KCl depolarization pulse is diminished in axonal growth cones of Munc13-1 KO motoneurons (*P = 0.0385, n = 27-29 cells, n = 4 cultures/mice for each genotype). \u003cstrong\u003eG\u003c/strong\u003e Graph indicates the percentage of failure response to membrane depolarization in control and Munc13-1 KO motoneurons (n = 4 cultures/mice for each genotype). Data are presented as mean ± SEM. *P ≤ 0.05 (Two-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/8277dbe440484171667775b8.png"},{"id":68730928,"identity":"fa9fba49-629a-4f9c-ac1a-657049b856e3","added_by":"auto","created_at":"2024-11-11 12:28:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":344773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAZ assembly is not altered in Munc13-2 KO motoneurons.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eWestern blot with total lysates from cultured motoneurons reveals reduced Munc13-2 protein levels after lentivirus transduction with shMunc13-2. \u003cstrong\u003eB\u003c/strong\u003eRepresentative images of cultured shCtrl- and shMunc13-2-transduced motoneurons, stained against Tau. \u003cstrong\u003eC\u003c/strong\u003e and \u003cstrong\u003eD\u003c/strong\u003e Graphs show that Munc13-2 downregulation does not affect axonal growth cone area (n = 36-45 cells), but impairs axon outgrowth (\u003cstrong\u003eD\u003c/strong\u003e: ****P \u0026lt; 0.0001, n = 211-215 cells) in cultured motoneurons (n = 3 cultures/mice). \u003cstrong\u003eE\u003c/strong\u003e Representative images of axonal growth cones of control and Munc13-2 KO motoneurons stained against β-actin. \u003cstrong\u003eF \u003c/strong\u003eand\u003cstrong\u003e G\u003c/strong\u003e The area of axonal growth cones (n = 52-53 cells), as well as the axon length (n = 131-136 cells), are comparable between control and Munc13-2 KO motoneurons (n = 3 cultures/mice for each genotype). \u003cstrong\u003eH \u003c/strong\u003eand\u003cstrong\u003e I\u003c/strong\u003e Representative images of axonal growth cones of control and Munc13-2 KO motoneurons stained against AZ proteins. \u003cstrong\u003eJ\u003c/strong\u003e-\u003cstrong\u003eN\u003c/strong\u003eMunc13-2 KO motoneurons exhibit unaltered levels of AZ components in axonal growth cones (n = 31-93 cells, n = 3 cultures/mice for each genotype). Data are presented as violin plots with the median shown as dashed lines. *P ≤ 0.05 (Two-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/dab3f0aa3ab12d020cecbb22.png"},{"id":68730194,"identity":"bdd7adbc-83fc-41b3-8358-70f74255dd70","added_by":"auto","created_at":"2024-11-11 12:20:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":202172,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLoss of Munc13-2 synaptic functions is compensated by Munc13-1. A \u003c/strong\u003eRepresentative images of axonal growth cones of motoneurons after transduction with a Munc13-1 overexpressing lentiviral construct (Munc13-1\u003csup\u003eOE\u003c/sup\u003e). \u003cstrong\u003eB \u003c/strong\u003eand\u003cstrong\u003e C\u003c/strong\u003e Graphs show a significant reduction in Munc13-2 levels in axonal growth cones (\u003cstrong\u003eB\u003c/strong\u003e: **P = 0.004, n = 60-62 cells), but unaltered levels in cell bodies of Munc13-1\u003csup\u003eOE\u003c/sup\u003e-transduced motoneurons (\u003cstrong\u003eC\u003c/strong\u003e: P = 0.247, n = 40 cells, n = 3 cultures/mice). \u003cstrong\u003eD\u003c/strong\u003e qRT-PCR shows unaltered mRNA levels of Munc13-2 in total lysates from Munc13-1\u003csup\u003eOE\u003c/sup\u003e-transduced motoneurons (n = 4 cultures/mice).\u003cstrong\u003e E\u003c/strong\u003e and \u003cstrong\u003eF\u003c/strong\u003e Representative Western blot and quantification show unaltered levels of Munc13-2 in total lysates from Munc13-1\u003csup\u003eOE\u003c/sup\u003e-transduced motoneurons (n = 6 cultures/mice).\u003cstrong\u003e G\u003c/strong\u003e Representative images of cell bodies of Munc13-1 KO motoneurons stained against Munc13-2. \u003cstrong\u003eH\u003c/strong\u003e Graph shows unaltered Munc13-2 levels in cell bodies of Munc13-1 KO motoneurons (P = 0.753, n = 62-63 cells, n = 3 cultures/mice for each genotype). \u003cstrong\u003eI\u003c/strong\u003e and \u003cstrong\u003eJ\u003c/strong\u003e Representative Western blot and quantification show unaltered levels of Mnc13-2 in total lysates from Munc13-1 KO motoneurons (n = 3 cultures/mice for each genotype). In \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and\u003cstrong\u003e H\u003c/strong\u003e, data are presented as violin plots with the median shown as dashed lines. *P ≤ 0.05 (Two-tailed Mann-Whitney U test). In \u003cstrong\u003eD\u003c/strong\u003e, \u003cstrong\u003eF \u003c/strong\u003eand \u003cstrong\u003eJ\u003c/strong\u003e data are presented as mean ± SEM. *P ≤ 0.05 (One-tailed Mann-Whitney U test).\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/4dfe43d59d6eba334d0460f1.png"},{"id":90344933,"identity":"a9caa163-dfff-4425-b3f7-3eae688f0359","added_by":"auto","created_at":"2025-09-01 16:08:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3430669,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/a618095e-9862-4bee-8b07-a5134d059def.pdf"},{"id":68729952,"identity":"e478d002-a68a-4fc9-b9d2-5aa5909ffc82","added_by":"auto","created_at":"2024-11-11 12:12:48","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2936099,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5305306/v1/47b302067f7a87b4dbdb8f4b.docx"}],"financialInterests":"","formattedTitle":"Loss of Synaptic Munc13-1 Underlies Neurotransmission Abnormalities in Spinal Muscular Atrophy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eImpaired synaptic function and degeneration are common pathological features of neurodegenerative diseases, including Spinal muscular atrophy (SMA) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SMA is the second most common fatal autosomal recessive genetic disease with an incidence of 1 per 6,000 births [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. SMA is caused by deletions of the Survival Motor Neuron 1 (\u003cem\u003eSMN1\u003c/em\u003e) gene [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. These mutations lead to degeneration of spinal motoneurons, loss of axons, denervation of neuromuscular junctions (NMJs), presynaptic accumulation of neurofilaments, endplate abnormalities, and muscle atrophy in SMA patients and animal models [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The SMN protein is required for the assembly of small nuclear ribonucleoproteins involved in pre-mRNA splicing [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] as well as regulation of axonal mRNA transport and local translation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Mouse models of SMA exhibit atrophic and smaller synapses, which are associated with impaired neurotransmitter release [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], disturbed clustering of voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channels (VGCCs) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and reduced evoked postsynaptic potential at neuromuscular endplates [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Motoneurons in SMA are believed to undergo degeneration in a \"dying-back\" manner, where the presynaptic terminal withdraws from the postsynaptic endplate, resulting in partially innervated endplates [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Additionally, the loss of axons in the ventral roots is observed to be more pronounced than the loss of motoneuron cell bodies, indicating a distal-to-proximal pattern of degeneration [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In SMA mice, NMJ defects manifest before the onset of clinical symptoms, suggesting that the NMJ represents an \"early pathological target\" in SMA [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the emerging evidence about synaptic impairments in SMA, the cellular mechanisms underlying synapse dysfunction and degeneration are not well understood.\u003c/p\u003e \u003cp\u003eNeurotransmitter release at the presynaptic membrane is a finely tuned process initiated by Ca\u003csup\u003e2+\u003c/sup\u003e entry through VGCCs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This occurs at specialized regions within the presynaptic plasma membrane known as active zones (AZs) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At the molecular level, AZs are densely populated with a complex array of proteins such as RIM, Munc13s, Piccolo (Pclo), and Bassoon (Bsn), which interact with synaptic vesicles (SVs) to orchestrate their docking, priming, and fusion with the plasma membrane [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. At the AZ within the presynaptic membranes, the Munc13 family release factor proteins achieve the temporal and spatial precision of SV release events in coordination with VGCCs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Munc13-1 plays a crucial role in synaptic plasticity by regulating SV priming and modifying the fusion competence of the readily releasable pool [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Munc13-1 KO mice exhibit severe neurological phenotypes including a complete failure of neurotransmitter release at central synapses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and NMJs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], leading to paralysis and early postnatal death [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In humans, \u003cem\u003eUNC13A\u003c/em\u003e mutations are associated with microcephaly, cortical hyperexcitability, and fatal myasthenia [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, we asked whether alterations in Munc13s synaptic functions might be associated with pathomechanisms underlying neurotransmission and plasticity defects in SMA.\u003c/p\u003e \u003cp\u003eHere, we elucidated the roles of Munc13-1 and Munc13-2 in neurotransmission in a mouse model of SMA and revealed that loss of Munc13-1, but not Munc13-2, is associated with synaptic aberrations in SMA mice. Our findings indicate that axonal localization of Munc13-1 and Munc13-2 mRNAs depends on Smn and is perturbed in SMA. Additionally, we show that loss of Munc13-1 leads to defective organization of the AZ and VGCCs in presynaptic membranes in motoneurons, contributing to impaired excitability. Together, these data demonstrate a role for Munc13-1 in AZ assembly and neurotransmitter release in motoneurons, highlighting a potential mechanism for synaptic abnormalities in SMA.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMice 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/6N/Smn1tm1Hung Tg(SMN2)2Hung/J line with C57BL/6J background [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. SMA litters (\u003cem\u003eSmn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003eHung\u003c/em\u003e\u003csup\u003e\u003cem\u003etg/+\u003c/em\u003e\u003c/sup\u003e, or Smn KO), and control litters (\u003cem\u003eSmn\u003c/em\u003e\u003csup\u003e\u003cem\u003e+/\u0026minus;\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003eHung\u003c/em\u003e\u003csup\u003e\u003cem\u003etg/+\u003c/em\u003e\u003c/sup\u003e or control) were derived from cross-breeding of Smn\u003csup\u003e+/\u0026minus;\u003c/sup\u003e to \u003cem\u003eSmn\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e,\u003cem\u003eHung\u003c/em\u003e\u003csup\u003e\u003cem\u003etg/tg\u003c/em\u003e\u003c/sup\u003e. C57BL/6J mice were used as WT for control experiments. Munc13-1 KO mice (\u003cem\u003eMunc13-1\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e)\u003c/em\u003e, and Munc13-2 KO mice \u003cem\u003e(Munc13-2\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] were obtained from Goettingen, Germany and cross-bred in-house.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEnrichment and culturing of primary mouse motoneurons\u003c/h3\u003e\n\u003cp\u003ePrimary mouse motoneurons were enriched via p75\u003csup\u003eNTR\u003c/sup\u003e antibody panning and cultured as previously described [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. For lentivirus transduction, cell suspensions were briefly exposed to lentiviral particles for 10 minutes 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 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Cells were grown onto glass coverslips for immunofluorescence, 24-well plates for Western blot and qRT-PCR, and \u0026micro;-dishes (Ibidi, 81156) for Ca\u003csup\u003e2+\u003c/sup\u003e imaging. Motoneuron culturing into compartmentalized microfluidic chambers was conducted as described earlier [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eImmunocytochemistry\u003c/h3\u003e\n\u003cp\u003eCells 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 \u0026micro;g/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\u0026deg;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\u0026deg;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-Ca\u003csup\u003e2+\u003c/sup\u003e channel N-type alpha-1B (Ca\u003csub\u003ev\u003c/sub\u003e2.2) (Synaptic System, 152305, 1:250), and mouse monoclonal anti-β-actin (GeneTex, GTX26276, 1:1000). Secondary antibodies are as followed: donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Alexa Fluor 488, Jackson ImmunoResearch, 715-545-150), donkey anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) AffiniPure (Alexa Fluor 488, Jackson ImmunoResearch, 711-545-152), donkey anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) AffiniPure (Cy3, Jackson ImmunoResearch, 711-165-152), and donkey anti-guinea pig IgG (H\u0026thinsp;+\u0026thinsp;L) AffiniPure (Cy5, Jackson ImmunoResearch, 706-175-148).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSingle-molecule fluorescence\u003c/b\u003e \u003cb\u003ein situ\u003c/b\u003e \u003cb\u003ehybridization (smFISH)\u003c/b\u003e\u003c/p\u003e \u003cp\u003esmFISH was conducted following the manufacturer\u0026rsquo;s instructions (ThermoFisher Scientific), as described earlier [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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\u0026deg;C overnight. Preamplifier, amplifier, and label probe oligonucleotides (diluted 1:25 in respective amplification buffers) were incubated each for 1 h at 40\u0026deg;C. Following the washing steps, cells were immunostained for Tau to visualize the neurite boundaries.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eFor 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 MgCl\u003csub\u003e2\u003c/sub\u003e, 2 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 20 mM glucose) and fixed with 4% PFA at 4\u0026deg;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\u0026deg;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-Ca\u003csup\u003e2+\u003c/sup\u003e channel P/Q-type (Ca\u003csub\u003ev\u003c/sub\u003e2.1) (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\u0026thinsp;+\u0026thinsp;L) AffiniPure (Cy3, Jackson ImmunoResearch, 711-165-152, 1:500), donkey anti-guinea pig IgG (H\u0026thinsp;+\u0026thinsp;L) AffiniPure (Cy5, Jackson ImmunoResearch, 706-175-148, 1:500).\u003c/p\u003e\n\u003ch3\u003eCa imaging and data quantification\u003c/h3\u003e\n\u003cp\u003eFor Ca\u003csup\u003e2+\u003c/sup\u003e imaging with cultured motoneurons, calcium indicator Oregon Green\u0026trade; 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 Ca\u003csup\u003e2+\u003c/sup\u003e imaging buffer (135 mM NaCl, 6 mM KCl, 1 mM MgCl\u003csub\u003e2\u003c/sub\u003e, 1 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 10 mM HEPES, and 5.5 mM glucose), Ca\u003csup\u003e2+\u003c/sup\u003e indicator (5 \u0026micro;M diluted in Ca\u003csup\u003e2+\u003c/sup\u003e imaging buffer) was added into cultured motoneuron dishes and incubated with for 15 min at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator. Residual calcium indicator dye was removed by twice washing with Ca\u003csup\u003e2+\u003c/sup\u003e imaging buffer and cells were imaged in 2 ml Ca\u003csup\u003e2+\u003c/sup\u003e 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\u0026times; 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\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e using a TOKAI HIT CO, LTD heated stage chamber. To monitor the spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e 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 \u0026micro;l of 90 mM KCl, followed by another minute of imaging. Images were taken at 16-bit with a resolution of 1.024 \u0026times; 1.024-pixel and a 2 \u0026times; 2 binning. Quantifications of Ca\u003csup\u003e2+\u003c/sup\u003e 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 \u0026ldquo;dynamic Z-axis profile\u0026rdquo;. The measured average intensities were normalized to the average intensities of the first 10 frames before a spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e spike appeared (F\u003csub\u003e0\u003c/sub\u003e) and plotted (F/F\u003csub\u003e0\u003c/sub\u003e). For KCl pulse experiments, the measured time-lapse intensities were normalized to the average of the first 20 frames immediately before KCl application (F\u003csub\u003e0\u003c/sub\u003e) and plotted (F/F\u003csub\u003e0\u003c/sub\u003e). BAR Plugin of Fiji was used for counting the Ca\u003csup\u003e2+\u003c/sup\u003e spikes.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and quantitative RT-PCR\u003c/b\u003e (\u003cb\u003eqRT-PCR)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor 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 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. 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.\u003c/p\u003e \u003cp\u003eFollowing primers were used for qRT-PCR: Gapdh (forward) 5\u0026rsquo;-AACTCCCACTCTTCCACCTTC-3\u0026rsquo; and (reverse) 5\u0026rsquo;-GGTCCAGGGTTTCTTACTCCTT-3\u0026rsquo;, Munc13-1 (forward) 5\u0026rsquo;-CACCACGCCCACCTACTGCTA-3\u0026rsquo; and (reverse) 5\u0026rsquo;-TTGCGCTCGCGGATCT-3\u0026prime;, Munc13-2 (forward) 5\u0026rsquo;-CTTGGCAGATGATAATGAGTA-3\u0026rsquo; and (reverse) 5\u0026rsquo;-GGTAGTCACTGTCTCGGTC-3\u0026prime;, Liprinα1 (forward) 5\u0026rsquo;-GATGGACTGCTTGACGGAAAC-3\u0026rsquo; and (reverse) 5\u0026rsquo;-GGCCATTGCTTCACGGAC-3\u0026prime;, Bsn (forward) 5\u0026rsquo;-GCTGCCAGCCAACCAG-3\u0026rsquo; and (reverse) 5\u0026rsquo;-CCACCAGGGAGGATCTTAGAG-3\u0026rsquo;, Pclo (forward) 5\u0026rsquo;-CCCGACCCATCCAAGGATATG-3\u0026rsquo; and (reverse) 5\u0026rsquo;-TGGTTGAATGCGGAGTTGCT-3\u0026rsquo;, and RIM2 (forward) 5\u0026rsquo;-CAGACCCTGGCTACTCCTGC-3\u0026rsquo; and (reverse) 5\u0026rsquo;-TACGGTGCTGGCAGTGTCTTG.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern blotting\u003c/h2\u003e \u003cp\u003eFor Western blotting of primary mouse motoneurons, 300,000 cells were plated and grown for 7 days. Cells were lysed directly in 1 \u0026times; 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\u0026deg;C for 5 min, briefly centrifuged, and loaded onto 4\u0026ndash;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 \u0026micro;g 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 \u0026micro;l cold sucrose lysis buffer (0.32 M sucrose, 5 mM HEPES, 1 \u0026times; protease inhibitor cocktail (Roche)) and centrifuged at 1000 \u0026times; g for 10 min at 4\u0026deg;C. Pellets (P1) were discarded and supernatants (S1) were centrifuged at 12000 \u0026times; g for 20 min at 4\u0026deg;C. Resulting supernatants (S2) were discarded and pellets (P2) containing crude synaptosomes were resuspended in 100 \u0026micro;l PBS. Protein concentration was measured and 20 \u0026micro;g total protein was loaded onto 4\u0026ndash;12% SDS gels. Primary antibodies were incubated overnight at 4\u0026deg;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), guinea pig polyclonal anti-Synaptophysin1 (Synaptic Systems, 101004, 1:5000), peroxidase AffiniPure donkey anti-goat IgG (H\u0026thinsp;+\u0026thinsp;L) (Biozol, 705-035-003, 1:10000), peroxidase AffiniPure donkey anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) (Biozol, 715-035-151, 1:10000), peroxidase AffiniPure goat anti-guinea pig IgG (H\u0026thinsp;+\u0026thinsp;L) (Jackson, 106-035-003, 1:10000), and peroxidase AffiniPure goat anti-rabbit IgG (H\u0026thinsp;+\u0026thinsp;L) (Biozol, 111-035-144, 1:10000).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlasmid cloning of shRNA constructs targeting Munc13s and lentivirus production\u003c/h3\u003e\n\u003cp\u003eFor 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\u0026reg; 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 [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The sequences of the antisense oligos used for shRNA cloning are as follows; Munc13-1: 5\u0026rsquo;-TCCCGTGTGAAACAAAGGT-3\u0026rsquo;, and Munc13-2: 5\u0026rsquo;-CGGAATAAACCAGAGATCT-3\u0026rsquo;. Lentiviruses were produced in HEK\u003csup\u003e293T\u003c/sup\u003e cells using TransIT-293 (Mirus, MIR2706) for transfection [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. pCMV-VSVG and pCMVΔR8.91 were employed as helper plasmids for the lentivirus production, and viral supernatants were collected by ultracentrifugation 60\u0026ndash;72 h after transfection. The virus titer was assessed in NSC\u003csup\u003e34\u003c/sup\u003e cells using serial dilutions.\u003c/p\u003e\n\u003ch3\u003eImage acquisition and processing\u003c/h3\u003e\n\u003cp\u003eImages were acquired with an Olympus Fluoview 1000 confocal microscope equipped with a 60\u0026times; 1.35-NA oil objective. For cultured motoneurons, 16-bit images with a resolution of 800 \u0026times; 800 pixels were taken from single z-stacks. For neuromuscular junctions (NMJs), confocal imaging involved 6 z-stacks at 0.5 \u0026micro;m 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\u0026times; 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\u0026trade; Microspheres, 0.2 \u0026micro;m, 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.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eTo 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. 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eFor 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\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM, or as violin plots, with the median shown as dashed lines. Statistical significance between two groups was assessed using the Mann-Whitney U test, while comparisons among multiple groups involved either the Mann-Whitney U test or one-way ANOVA followed by the Kruskal-Wallis test with Dunn\u0026rsquo;s Multiple Comparison post-hoc test.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eTranscripts for Munc13s, and Liprinα1, but not Pclo, Bsn, and RIM are enriched in motor axons\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRNA sequencing studies have shown that transcriptomes of neuronal processes, dendrites [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], and axons [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] include transcripts encoding synapse-related proteins. The Smn protein plays a key role in axonal mRNA transport and local translation of various targets [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and the disruption of these functions is linked to impaired synaptic transmission in SMA [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. This raises the possibility that improper mRNA localization of AZ components may contribute to synapse dysfunction in SMA.\u003c/p\u003e \u003cp\u003eTo explore this hypothesis, we investigated the localization of transcripts for AZ proteins within cultured mouse motoneurons using compartmentalized microfluidic chambers (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Quantitative real-time PCR (qRT-PCR) analysis revealed a significant enrichment of transcripts for the AZ proteins including Munc13-1, and Liprinα1 in distal axons compared to the somatodendritic compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Although Munc13-2 transcripts also showed enrichment in axons, this was not statistically significant (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In contrast, transcripts for Pclo were significantly enriched in the somatodendritic compartment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). To further validate these findings, we employed single-molecule fluorescence in situ hybridization (smFISH) to visualize the subcellular localization of these transcripts. The smFISH results confirmed the localization of Munc13-1, Munc13-2, and Liprinα1 transcripts in distal axons in cultured motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eThe assembly of the presynaptic active zone is diminished in Smn KO motoneurons\u003c/h2\u003e \u003cp\u003eNext, we assessed the impact of Smn loss of function on the axonal localization of transcripts for AZ proteins in cultured spinal motoneurons. To this end we employed a previously described and validated shRNA lentiviral construct to specifically target the mouse Smn [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Following Smn knockdown, qRT-PCR analysis demonstrated a significant reduction in the levels of Munc13-1, Munc13-2, and Liprinα1 transcripts in axons of cultured motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). These data suggest that Smn is crucial for the proper mRNA localization of these AZ components in motor axons. Since Smn is also involved in mRNA splicing, we investigated whether the loss of Smn affects the overall expression of AZ proteins. qRT-PCR analysis revealed a slight but statistically significant reduction in the total expression of AZ components in the brains of Smn KO mice, while no significant changes were observed in the spinal cord (Fig. S1A). These slight alterations, however, could be attributed to the severe synaptic loss observed in the late stages of SMA using P10 Smn KO mice. To evaluate this possibility, we analyzed the total expression of Munc13 proteins in cultured motoneurons from E13 Smn KO mice using qRT-PCR. The results showed no significant reduction in Munc13-1 and Munc13-2 mRNA levels in Smn KO motoneurons at embryonic stages (Fig. S1B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe then examined the distribution and levels of key AZ proteins in cultured motoneurons derived from Smn KO mice. Immunostaining revealed a marked reduction in Munc13-1 and Munc13-2 protein levels in Smn KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D). Importantly, these reductions were observed specifically in axonal growth cones, with no significant changes detected in cell bodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, D). To confirm these findings, we conducted Western blot assays on total lysates from cultured Smn KO motoneurons, as well as brain and spinal cord tissues from Smn KO mice. The results showed a minor reduction in Munc13-1 protein levels in cultured Smn KO motoneurons (Fig. S1C), but no significant changes in its levels in brain and spinal cord tissues (Fig. S1D, E). In contrast, Munc13-2 protein levels were elevated in total lysates from cultured Smn KO motoneurons (Fig. S1F), as well as in brain (Fig. S1G) and spinal cord tissues (Fig. S1H) of Smn KO mice, suggesting a compensatory upregulation mechanism.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of other AZ components including Pclo, RIM1/2, Bsn, and Liprinα1 revealed a broader disruption in AZ protein organization within the growth cones of Smn KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-J). Additionally, we assessed the levels of Munc13 isoforms at NMJs from the transverse abdominis muscles (TVA) of Smn KO mice. Notably, we observed a significant reduction in Munc13-1 levels at NMJs, while Munc13-2 levels remained unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D). The specificity of the Munc13-1 and Munc13-2 antibodies was validated by Western blot analysis of synaptosome fractions from the cortices of Munc13s KO mice (Fig. S2A, B), as well as by immunohistochemistry analysis of NMJs from Munc13s KO mice (Fig. S2C, D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThese findings suggest that loss of Smn severely impairs the organization of AZ components within the presynaptic membranes, in particular Munc13-1. This disruption likely contributes to the synaptic abnormalities seen in SMA, underscoring the vulnerability of the synaptic assembly process in the absence of a functional Smn protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eMunc13-1 tethers VGCCs into presynaptic AZs in motoneurons\u003c/h2\u003e \u003cp\u003eAt presynaptic AZs, Munc13-1 interacts with proteins like RIM and RIM binding protein (RIM-BP), forming a complex that helps tether SVs close to VGCCs [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This precise spatial alignment between Munc13s and VGCCs ensures temporal and spatial regulation of neurotransmitter release [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. To gain a detailed view of the spatial colocalization between Munc13 isoforms and Ca\u003csub\u003ev\u003c/sub\u003e2.2 (a subtype of VGCCs) in axonal growth cones of wildtype (WT) motoneurons, we employed Structured Illumination Microscopy (SIM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Quantitative analysis revealed that VGCCs (Ca\u003csub\u003ev\u003c/sub\u003e2.2) colocalize significantly more with Munc13-1 than Munc13-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). This finding is consistent with previous studies that demonstrated a tighter coupling between Unc13a and VGCCs at AZs in \u003cem\u003eDrosophila\u003c/em\u003e NMJs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Intriguingly, SIM imaging showed reduced colocalization between Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.2 at AZs in cultured Smn KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H). This reduction indicates a disruption in the spatial organization of these critical synaptic components. Similarly, confocal imaging of NMJs from the TVA muscles of Smn KO and control mice showed a significant decrease in the colocalization between Munc13-1 and Ca\u003csub\u003ev\u003c/sub\u003e2.1 (another VGCC subtype) at NMJs in Smn KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI, J). These findings suggest that the loss of Smn disrupts the spatial organization of Munc13-1 and VGCCs at both axonal growth cones and NMJs, potentially contributing to the synaptic dysfunction in SMA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDepletion of Munc13-1 affects the AZ assembly and diminishes neuronal excitability\u003c/h2\u003e \u003cp\u003eWe investigated the role of Munc13-1 and Munc13-2 in motoneuron development and function by employing shRNA constructs to selectively knock down these proteins. Additionally, we analyzed the phenotypes of cultured motoneurons obtained from Munc13s KO mice. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Western blot analysis confirmed effective knockdown of Munc13-1 in motoneurons transduced with shRNA targeting Munc13-1. Imaging of axonal growth cones revealed that Munc13-1-depleted motoneurons exhibited significantly smaller growth cones compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C), suggesting impaired growth cone differentiation. Despite this reduction in growth cone size, axon outgrowth was unaffected by Munc13-1 depletion (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Interestingly, measurements of growth cone area and axon length in Munc13-1 KO motoneurons were comparable to those in control motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE-G). This discrepancy may be due to compensatory mechanisms activated in Munc13-1 KO motoneurons. Furthermore, to elucidate whether Munc13-1 loss recapitulates SMA phenotype, we examined the AZ components and VGCCs in Munc13-1 KO motoneurons by immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, K). These quantitative analyses showed that Ca\u003csub\u003ev\u003c/sub\u003e2.2 levels were reduced in axonal growth cones of Munc13-1 KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, I), while Munc13-2 levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, J). In line with this, the levels of RIM1/2, Pclo, and Bsn were reduced in axonal growth cones of Munc13-1 KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eK-N). Intriguingly, these abnormalities in AZ assembly and VGCC clustering correlated with impaired excitability in Munc13-1 KO motoneurons. As demonstrated by Ca\u003csup\u003e2+\u003c/sup\u003e imaging, Munc13-1 KO motoneurons exhibit reduced spontaneous Ca\u003csup\u003e2+\u003c/sup\u003e transients in growth cones (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B), although the amplitude of these transients was not altered (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). Additionally, we applied a KCl depolarization to measure the evoked Ca\u003csup\u003e2+\u003c/sup\u003e transient response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). The maximum response to induced depolarization was diminished in growth cones of Munc13-1 KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, F), and the percentage of failed responses to membrane depolarization was higher (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In summary, these findings indicate that depletion of Munc13-1 leads to defects in growth cone differentiation, impaired AZ assembly, and abnormal VGCC clustering, which together contribute to reduced neuronal excitability in motoneurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eMunc13-2 synaptic functions are compensated by Munc13-1\u003c/h2\u003e \u003cp\u003eTo assess the role of Munc13-2 in motoneurons, we knocked down Munc13-2 using lentivirus transduction and analyzed its subsequent effects on AZ assembly and axonal growth and differentiation. Western blot analysis confirmed a reduction in Munc13-2 protein levels in motoneurons transduced with shRNA targeting Munc13-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Downregulation of Munc13-2 did not affect the area of axonal growth cones but impaired axon outgrowth in cultured motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-D). Next, we analyzed the axon growth and differentiation in cultured Munc13-2 KO motoneurons. Our data revealed that the area of axonal growth cones and the axon length of Munc13-2 KO motoneurons were comparable to those of the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-G). Moreover, immunostaining of AZ proteins revealed unaltered levels of AZ components in axonal growth cones of cultured Munc13-2 KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eH-N), suggesting that Munc13-2 is not essential for AZ assembly or growth cone differentiation in spinal motoneurons.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo explore potential compensatory mechanisms for Munc13-2 loss, we analyzed the effects of Munc13-1 overexpression in motoneurons. For this purpose, we generated a lentiviral construct expressing Munc13-1 (Munc13-1\u003csup\u003eOE\u003c/sup\u003e) and confirmed its overexpression in cultured motoneurons through immunostaining and Western blot assays (Fig. S3A-C). Immunostaining revealed that upon transduction of motoneurons with Munc13-1\u003csup\u003eOE\u003c/sup\u003e virus, Munc13-2 levels decreased in axonal growth cones, while its levels remained unaltered in the soma (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). To further verify that Munc13-1 overexpression does not alter the total expression of Munc13-2, we conducted qRT-PCR and Western blot. The obtained data revealed no significant changes in Munc13-2 mRNA levels in total lysates from Munc13-1\u003csup\u003eOE\u003c/sup\u003e-transduced motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). This indicates that the specific reduction in Munc13-2 protein in axonal growth cones was not due to its transcriptional downregulation. Western blot analysis further confirmed that overall protein levels of Munc13-2 remain unaltered by Munc13-1 overexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, F). Interestingly, unlike Munc13-2, overexpression of Munc13-1 in motoneurons led to increased levels of Ca\u003csub\u003ev\u003c/sub\u003e2.2 in axonal growth cones (Fig. S3D) without affecting the overall levels of other AZ proteins (Fig. S3E-H). These data unravel a specific and non-redundant role for Munc13-1 in VGCC tethering.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether a similar compensatory mechanism occurs for Munc13-1 loss through Munc13-2, we measured the Munc13-2 levels in cultured Munc13-1 KO motoneurons. Notably, we observed that Munc13-2 levels remained unchanged in axonal growth cones of Munc13-1 KO motoneurons (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH, J). Consistent with this finding, total Munc13-2 levels were not elevated in Munc13-1 KO motoneurons, as demonstrated by immunostaining of cell bodies and Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eG-J).\u003c/p\u003e \u003cp\u003eIn conclusion, while Munc13-2 downregulation affects axon growth, it does not disrupt AZ assembly or growth cone differentiation. Furthermore, these data suggest that Munc13-1 has redundant functions with Munc13-2 and can compensate for Munc13-2 in motoneurons, thereby maintaining synaptic functions despite the loss of Munc13-2.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study provides new insights into the role of Munc13 proteins in the pathogenesis of synaptic dysfunctions in SMA. Our findings reveal that Munc13-1, but not Munc13-2, plays a critical role in the organization of AZs and the clustering of VGCCs in motoneurons, suggesting that Munc13-1 dysregulation contributes to the synaptic defects observed in SMA.\u003c/p\u003e \u003cp\u003eA common therapeutic strategy for SMA involves increasing SMN protein levels through gene therapy approaches, referred to as SMN-based therapies. These treatments target the \u003cem\u003eSMN2\u003c/em\u003e gene to enhance SMN protein production [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, non-responsive cases often arise due to two main factors: patients who miss the therapeutic window benefit less from SMN-repletion therapies, and there is still a significant knowledge gap in understanding how SMN deficiency leads to NMJ dysfunction and synapse degeneration. Understanding the cellular mechanisms driving synaptic deficiencies is therefore critical for identifying disease modifiers and developing combination therapies to improve patient outcomes. Our findings indicate that Munc13-1 depletion in motoneurons results in aberrant assembly of presynaptic AZs and impairs clustering of VGCCs in axonal growth cones, leading to reduced neuronal excitability. This is consistent with previous studies highlighting the Munc13-1 ability to tether VGCCs to SVs [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and defective neurotransmitter release in its absence in hippocampal neurons and at neuromuscular junctions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In line with this, we observed reduced Munc13-1 levels in presynaptic terminals of cultured Smn KO motoneurons and at NMJs in Smn KO mice. These findings provide a molecular mechanism that links Smn deficiency to synaptic abnormalities in SMA.\u003c/p\u003e \u003cp\u003eInterestingly, although Munc13-2 also localizes to axonal growth cones, its role appears to be less crucial in motoneurons. Our data show that the loss of Munc13-2 does not significantly affect AZ assembly or growth cone differentiation, suggesting that Munc13-1 can compensate for the absence of Munc13-2. Conversely, in Munc13-1 KO motoneurons, the Munc13-2 levels remained unchanged, suggesting that Munc13-1 cannot be fully compensated by Munc13-2 in motoneurons. This lack of compensation may contribute to the synaptic deficits observed in SMA, emphasizing the non-redundant function of Munc13-1 in maintaining synaptic function. Moreover, the failure to upregulate Munc13-2 in response to Munc13-1 loss suggests that therapeutic strategies aimed at increasing Munc13-2 expression may not be sufficient to restore synaptic function in SMA.\u003c/p\u003e \u003cp\u003eIn neurons, subcellular mRNA localization and intra-axonal local translation are conserved cellular mechanisms that regulate synaptogenesis, plasticity, and axon regeneration through temporal and spatial modulation of the local proteome [\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Disruption in local translation impacts neuronal function and viability, leading to neurodegeneration in ALS, SMA, Fragile X Syndrome, Alzheimer's disease, and Parkinson's disease [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Local translation is particularly crucial for spinal motoneurons due to their long axons and their unique anatomical and morphological characteristics [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. These distinct features make motoneurons especially vulnerable to degeneration resulting from disruptions in protein synthesis, particularly within the axon. Impaired local translation in SMA is attributed to disrupted mRNA subcellular localization [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and the loss of direct interaction between the SMN protein and ribosomes, which is essential for the recruitment of mRNA onto polysomes [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Moreover, loss of SMN is associated with disturbed ribosome assembly and impaired formation of the rough endoplasmic reticulum (RER) in presynaptic compartments in motoneurons, leading to delayed translation initiation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Here, we demonstrate that Smn downregulation results in a significant reduction in Munc13-1 and Liprinα1 mRNAs in axons of primary cultured mouse motoneurons. This finding highlights the importance of mRNA transport and local translation of synaptic proteins for the maintenance of synaptic integrity. This aligns with previous reports that Smn plays a critical role in mRNA localization and that its deficiency leads to widespread defects in axonal protein synthesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The mechanisms by which Smn regulates the axonal translocation of Munc13-1 mRNA in motoneurons may involve the RNA-binding protein heterogeneous nuclear ribonucleoprotein R (hnRNP R). Importantly, iCLIP assays have revealed that Munc13-1 mRNA binds to hnRNP R via its 3' untranslated region (3'UTR) [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. This interaction suggests that the correct axonal localization of Munc13-1 depends on the presence of hnRNP R within axons. hnRNP R is a binding partner of Smn, and in Smn KO motoneurons, axonal levels of hnRNP R are reduced [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Consequently, reduced levels of hnRNP R in axons of Smn KO motoneurons may disrupt the localization of Munc13-1 mRNAs within axons, potentially affecting axonal function.\u003c/p\u003e \u003cp\u003eThus, to fully restore synaptic function in SMA, therapies targeting SMN function may need to focus as well on correcting the mRNA transport and local translation processes. In conclusion, our findings demonstrate that Munc13-1 is pivotal for AZ assembly and neurotransmitter release in motoneurons, and its dysregulation contributes to the synaptic defects observed in SMA. The inability of Munc13-2 to fully compensate for Munc13-1 loss underscores the non-redundant functions of these proteins in maintaining synaptic integrity. These insights provide a deeper understanding of the molecular mechanisms underlying synaptic dysfunction in SMA and may inform the development of targeted therapies aimed at mitigating synaptic loss in this devastating disease.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Markus Sauer for his support with SIM, Michael Briese and Kilian Katzenberger for their technical assistance, Regine Sendtner for her work in animal breeding, and Hildegard Troll for her contribution to lentivirus production.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by DFG, Grant Se697/7-1 to Michael Sendtner.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM. M. and M. S. conceived the project, designed the experiments, and prepared the manuscript. M. M. and CC. D. conducted the experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll datasets are provided within the main text or Supplementary Materials and can be obtained from the corresponding authors upon request. This study includes no data deposited in external repositories.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was performed in line with the principles of the Declaration of Germany. Approval was granted by the Ethics Committee of the University of Wuerzburg.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBae, J.R. and S.H. Kim, \u003cem\u003eSynapses in neurodegenerative diseases.\u003c/em\u003e BMB Rep, 2017. \u003cstrong\u003e50\u003c/strong\u003e(5): p. 237-246.\u003c/li\u003e\n\u003cli\u003eWilson, D.M., 3rd, et al., \u003cem\u003eHallmarks of neurodegenerative diseases.\u003c/em\u003e Cell, 2023. \u003cstrong\u003e186\u003c/strong\u003e(4): p. 693-714.\u003c/li\u003e\n\u003cli\u003eCrawford, T.O. and C.A. Pardo, \u003cem\u003eThe neurobiology of childhood spinal muscular atrophy.\u003c/em\u003e Neurobiol Dis, 1996. \u003cstrong\u003e3\u003c/strong\u003e(2): p. 97-110.\u003c/li\u003e\n\u003cli\u003eGoulet, B.B., R. Kothary, and R.J. Parks, \u003cem\u003eAt the \u0026quot;junction\u0026quot; of spinal muscular atrophy pathogenesis: the role of neuromuscular junction dysfunction in SMA disease progression.\u003c/em\u003e Curr Mol Med, 2013. \u003cstrong\u003e13\u003c/strong\u003e(7): p. 1160-74.\u003c/li\u003e\n\u003cli\u003eTisdale, S. and L. Pellizzoni, \u003cem\u003eDisease mechanisms and therapeutic approaches in spinal muscular atrophy.\u003c/em\u003e J Neurosci, 2015. \u003cstrong\u003e35\u003c/strong\u003e(23): p. 8691-700.\u003c/li\u003e\n\u003cli\u003eWee, C.D., L. Kong, and C.J. Sumner, \u003cem\u003eThe genetics of spinal muscular atrophies.\u003c/em\u003e Curr Opin Neurol, 2010. \u003cstrong\u003e23\u003c/strong\u003e(5): p. 450-8.\u003c/li\u003e\n\u003cli\u003ePillai, R.S., et al., \u003cem\u003eUnique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.\u003c/em\u003e Genes Dev, 2003. \u003cstrong\u003e17\u003c/strong\u003e(18): p. 2321-33.\u003c/li\u003e\n\u003cli\u003eFallini, C., G.J. Bassell, and W. Rossoll, \u003cem\u003eSpinal muscular atrophy: the role of SMN in axonal mRNA regulation.\u003c/em\u003e Brain Res, 2012. \u003cstrong\u003e1462\u003c/strong\u003e: p. 81-92.\u003c/li\u003e\n\u003cli\u003eMoradi, M., et al., \u003cem\u003eDifferential roles of alpha-, beta-, and gamma-actin in axon growth and collateral branch formation in motoneurons.\u003c/em\u003e J Cell Biol, 2017. \u003cstrong\u003e216\u003c/strong\u003e(3): p. 793-814.\u003c/li\u003e\n\u003cli\u003eKong, L., et al., \u003cem\u003eImpaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice.\u003c/em\u003e J Neurosci, 2009. \u003cstrong\u003e29\u003c/strong\u003e(3): p. 842-51.\u003c/li\u003e\n\u003cli\u003eJablonka, S., et al., \u003cem\u003eDefective Ca2+ channel clustering in axon terminals disturbs excitability in motoneurons in spinal muscular atrophy.\u003c/em\u003e J Cell Biol, 2007. \u003cstrong\u003e179\u003c/strong\u003e(1): p. 139-49.\u003c/li\u003e\n\u003cli\u003eXu, Y., et al., \u003cem\u003eDefects in Neuromuscular Transmission May Underlie Motor Dysfunction in Spinal and Bulbar Muscular Atrophy.\u003c/em\u003e J Neurosci, 2016. \u003cstrong\u003e36\u003c/strong\u003e(18): p. 5094-106.\u003c/li\u003e\n\u003cli\u003eMurray, L.M., et al., \u003cem\u003eSelective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy.\u003c/em\u003e Hum Mol Genet, 2008. \u003cstrong\u003e17\u003c/strong\u003e(7): p. 949-62.\u003c/li\u003e\n\u003cli\u003eCourtney, N.L., et al., \u003cem\u003eReduced P53 levels ameliorate neuromuscular junction loss without affecting motor neuron pathology in a mouse model of spinal muscular atrophy.\u003c/em\u003e Cell Death Dis, 2019. \u003cstrong\u003e10\u003c/strong\u003e(7): p. 515.\u003c/li\u003e\n\u003cli\u003eBohme, M.A., et al., \u003cem\u003eActive zone scaffolds differentially accumulate Unc13 isoforms to tune Ca(2+) channel-vesicle coupling.\u003c/em\u003e Nat Neurosci, 2016. \u003cstrong\u003e19\u003c/strong\u003e(10): p. 1311-20.\u003c/li\u003e\n\u003cli\u003eS\u0026uuml;dhof, T.C., \u003cem\u003eThe presynaptic active zone.\u003c/em\u003e Neuron, 2012. \u003cstrong\u003e75\u003c/strong\u003e(1): p. 11-25.\u003c/li\u003e\n\u003cli\u003eAugustin, I., et al., \u003cem\u003eMunc13-1 is essential for fusion competence of glutamatergic synaptic vesicles.\u003c/em\u003e Nature, 1999. \u003cstrong\u003e400\u003c/strong\u003e(6743): p. 457-61.\u003c/li\u003e\n\u003cli\u003eVaroqueaux, F., et al., \u003cem\u003eAberrant morphology and residual transmitter release at the Munc13-deficient mouse neuromuscular synapse.\u003c/em\u003e Mol Cell Biol, 2005. \u003cstrong\u003e25\u003c/strong\u003e(14): p. 5973-84.\u003c/li\u003e\n\u003cli\u003eVaroqueaux, F., et al., \u003cem\u003eTotal arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming.\u003c/em\u003e Proc Natl Acad Sci U S A, 2002. \u003cstrong\u003e99\u003c/strong\u003e(13): p. 9037-42.\u003c/li\u003e\n\u003cli\u003eEngel, A.G., et al., \u003cem\u003eLoss of MUNC13-1 function causes microcephaly, cortical hyperexcitability, and fatal myasthenia.\u003c/em\u003e Neurol Genet, 2016. \u003cstrong\u003e2\u003c/strong\u003e(5): p. e105.\u003c/li\u003e\n\u003cli\u003eHsieh-Li, H.M., et al., \u003cem\u003eA mouse model for spinal muscular atrophy.\u003c/em\u003e Nat Genet, 2000. \u003cstrong\u003e24\u003c/strong\u003e(1): p. 66-70.\u003c/li\u003e\n\u003cli\u003eDeng, C., et al., \u003cem\u003eDynamic remodeling of ribosomes and endoplasmic reticulum in axon terminals of motoneurons.\u003c/em\u003e J Cell Sci, 2021. \u003cstrong\u003e134\u003c/strong\u003e(22).\u003c/li\u003e\n\u003cli\u003eWiese, S., et al., \u003cem\u003eIsolation and enrichment of embryonic mouse motoneurons from the lumbar spinal cord of individual mouse embryos.\u003c/em\u003e Nat Protoc, 2010. \u003cstrong\u003e5\u003c/strong\u003e(1): p. 31-8.\u003c/li\u003e\n\u003cli\u003eChand, K.K., et al., \u003cem\u003eLoss of \u003c/em\u003e\u003cem\u003e\u0026beta;\u003c/em\u003e\u003cem\u003e2-laminin alters calcium sensitivity and voltage-gated calcium channel maturation of neurotransmission at the neuromuscular junction.\u003c/em\u003e J Physiol, 2015. \u003cstrong\u003e593\u003c/strong\u003e(1): p. 245-65.\u003c/li\u003e\n\u003cli\u003eSaal, L., et al., \u003cem\u003eSubcellular transcriptome alterations in a cell culture model of spinal muscular atrophy point to widespread defects in axonal growth and presynaptic differentiation.\u003c/em\u003e RNA, 2014. \u003cstrong\u003e20\u003c/strong\u003e(11): p. 1789-802.\u003c/li\u003e\n\u003cli\u003eSubramanian, N., et al., \u003cem\u003eRole of Na(v)1.9 in activity-dependent axon growth in motoneurons.\u003c/em\u003e Hum Mol Genet, 2012. \u003cstrong\u003e21\u003c/strong\u003e(16): p. 3655-67.\u003c/li\u003e\n\u003cli\u003eRehberg, M., et al., \u003cem\u003eA new non-disruptive strategy to target calcium indicator dyes to the endoplasmic reticulum.\u003c/em\u003e Cell Calcium, 2008. \u003cstrong\u003e44\u003c/strong\u003e(4): p. 386-99.\u003c/li\u003e\n\u003cli\u003eHobson, B.D., et al., \u003cem\u003eSubcellular and regional localization of mRNA translation in midbrain dopamine neurons.\u003c/em\u003e Cell Rep, 2022. \u003cstrong\u003e38\u003c/strong\u003e(2): p. 110208.\u003c/li\u003e\n\u003cli\u003eCajigas, I.J., et al., \u003cem\u003eThe local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging.\u003c/em\u003e Neuron, 2012. \u003cstrong\u003e74\u003c/strong\u003e(3): p. 453-66.\u003c/li\u003e\n\u003cli\u003eHennlein, L., et al., \u003cem\u003ePlastin 3 rescues cell surface translocation and activation of TrkB in spinal muscular atrophy.\u003c/em\u003e J Cell Biol, 2023. \u003cstrong\u003e222\u003c/strong\u003e(3).\u003c/li\u003e\n\u003cli\u003eTushev, G., et al., \u003cem\u003eAlternative 3\u0026apos; UTRs Modify the Localization, Regulatory Potential, Stability, and Plasticity of mRNAs in Neuronal Compartments.\u003c/em\u003e Neuron, 2018. \u003cstrong\u003e98\u003c/strong\u003e(3): p. 495-511.e6.\u003c/li\u003e\n\u003cli\u003eKong, L., et al., \u003cem\u003eImpaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice.\u003c/em\u003e J Neurosci, 2009. \u003cstrong\u003e29\u003c/strong\u003e(3): p. 842-51.\u003c/li\u003e\n\u003cli\u003eJablonka, S. and M. Sendtner, \u003cem\u003eDevelopmental regulation of SMN expression: pathophysiological implications and perspectives for therapy development in spinal muscular atrophy.\u003c/em\u003e Gene Ther, 2017. \u003cstrong\u003e24\u003c/strong\u003e(9): p. 506-513.\u003c/li\u003e\n\u003cli\u003eDittman, J.S., \u003cem\u003eUnc13: a multifunctional synaptic marvel.\u003c/em\u003e Curr Opin Neurobiol, 2019. \u003cstrong\u003e57\u003c/strong\u003e: p. 17-25.\u003c/li\u003e\n\u003cli\u003eHolt, C.E., K.C. Martin, and E.M. Schuman, \u003cem\u003eLocal translation in neurons: visualization and function.\u003c/em\u003e Nat Struct Mol Biol, 2019. \u003cstrong\u003e26\u003c/strong\u003e(7): p. 557-566.\u003c/li\u003e\n\u003cli\u003eKang, H. and E.M. Schuman, \u003cem\u003eA requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity.\u003c/em\u003e Science, 1996. \u003cstrong\u003e273\u003c/strong\u003e(5280): p. 1402-6.\u003c/li\u003e\n\u003cli\u003eJung, H., et al., \u003cem\u003eRemote control of gene function by local translation.\u003c/em\u003e Cell, 2014. \u003cstrong\u003e157\u003c/strong\u003e(1): p. 26-40.\u003c/li\u003e\n\u003cli\u003eLin, J.Q., F.W. van Tartwijk, and C.E. Holt, \u003cem\u003eAxonal mRNA translation in neurological disorders.\u003c/em\u003e RNA Biol, 2021. \u003cstrong\u003e18\u003c/strong\u003e(7): p. 936-961.\u003c/li\u003e\n\u003cli\u003eShigeoka, T., et al., \u003cem\u003eDynamic Axonal Translation in Developing and Mature Visual Circuits.\u003c/em\u003e Cell, 2016. \u003cstrong\u003e166\u003c/strong\u003e(1): p. 181-92.\u003c/li\u003e\n\u003cli\u003eRagagnin, A.M.G., et al., \u003cem\u003eMotor Neuron Susceptibility in ALS/FTD.\u003c/em\u003e Front Neurosci, 2019. \u003cstrong\u003e13\u003c/strong\u003e: p. 532.\u003c/li\u003e\n\u003cli\u003eBriese, M., et al., \u003cem\u003eLoss of Tdp-43 disrupts the axonal transcriptome of motoneurons accompanied by impaired axonal translation and mitochondria function.\u003c/em\u003e Acta Neuropathol Commun, 2020. \u003cstrong\u003e8\u003c/strong\u003e(1): p. 116.\u003c/li\u003e\n\u003cli\u003eLauria, F., et al., \u003cem\u003eSMN-primed ribosomes modulate the translation of transcripts related to spinal muscular atrophy.\u003c/em\u003e Nat Cell Biol, 2020. \u003cstrong\u003e22\u003c/strong\u003e(10): p. 1239-1251.\u003c/li\u003e\n\u003cli\u003eDeng, C., et al., \u003cem\u003eImpaired dynamic interaction of axonal endoplasmic reticulum and ribosomes contributes to defective stimulus-response in spinal muscular atrophy.\u003c/em\u003e Transl Neurodegener, 2022. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 31.\u003c/li\u003e\n\u003cli\u003eRossoll, W., et al., \u003cem\u003eSmn, the spinal muscular atrophy-determining gene product, modulates axon growth and localization of beta-actin mRNA in growth cones of motoneurons.\u003c/em\u003e J Cell Biol, 2003. \u003cstrong\u003e163\u003c/strong\u003e(4): p. 801-12.\u003c/li\u003e\n\u003cli\u003eBriese, M., et al., \u003cem\u003ehnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons.\u003c/em\u003e Proc Natl Acad Sci U S A, 2018. \u003cstrong\u003e115\u003c/strong\u003e(12): p. E2859-E2868.\u003c/li\u003e\n\u003cli\u003eRossoll, W., et al., \u003cem\u003eSpecific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons?\u003c/em\u003e Hum Mol Genet, 2002. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 93-105.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Active Zone Assembly, Axonal mRNA Transport, Munc13s, Spinal Muscular atrophy, Synapse Degeneration","lastPublishedDoi":"10.21203/rs.3.rs-5305306/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5305306/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpinal muscular atrophy (SMA) is a devastating neurodegenerative disease characterized by degeneration of spinal motoneurons, leading to muscle atrophy and synaptic loss. SMN functions in mRNA splicing, transport, and local translation are crucial for maintaining synaptic integrity. Within the presynaptic membrane, the active zone orchestrates the docking and priming of synaptic vesicles. The Munc13 family proteins are key active zone components that operate precise neurotransmitter release in conjunction with voltage-gated Ca\u003csup\u003e2+\u003c/sup\u003e channels (VGCCs). However, the role of Munc13s in synaptic dysfunction in SMA remains elusive. Our findings reveal that Munc13-1 loss, but not Munc13-2, is closely linked to synaptic aberrations in SMA. Specifically, Munc13-1 mRNA localization in axons is dependent on Smn, and its disruption leads to impaired AZ assembly and VGCC clustering in motoneurons, ultimately reducing neuronal excitability. In contrast, Munc13-2 does not appear to be essential for AZ assembly or motoneuron differentiation, as its functions can be compensated by Munc13-1. These findings highlight the pivotal role of Munc13-1 in synapse integrity and point to potential therapeutic targets for mitigating synaptic loss in SMA.\u003c/p\u003e","manuscriptTitle":"Loss of Synaptic Munc13-1 Underlies Neurotransmission Abnormalities in Spinal Muscular Atrophy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-11 12:12:43","doi":"10.21203/rs.3.rs-5305306/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-12-18T05:54:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-10-31T17:41:29+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-30T20:46:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-28T10:17:31+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2024-10-21T10:27:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f42ab3ad-cf96-45d4-9711-8cddbf9bc990","owner":[],"postedDate":"November 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-01T16:03:40+00:00","versionOfRecord":{"articleIdentity":"rs-5305306","link":"https://doi.org/10.1007/s00018-025-05859-7","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-08-29 15:58:20","publishedOnDateReadable":"August 29th, 2025"},"versionCreatedAt":"2024-11-11 12:12:43","video":"","vorDoi":"10.1007/s00018-025-05859-7","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05859-7","workflowStages":[]},"version":"v1","identity":"rs-5305306","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5305306","identity":"rs-5305306","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}