Introduction

The vertebrate neuromuscular junction (NMJ) is a peripheral cholinergic synapse formed by a motor axon terminal and a skeletal muscle fiber specialization, a structure capped by terminal Schwann cells. During embryonic NMJ development, pre and postsynaptic signals regulate both the clustering of acetylcholine receptors (AChRs) on the muscle membrane and the subsequent innervation of nascent postsynaptic domains. During NMJ maturation, early post-natal elliptical postsynaptic plaques gradually re-organize to form pretzel-like structures, complex arrangements containing regions of high and low AChR density [5, 46, 54]. At the ultrastructural level, AChRs concentrate at the edges of secondary folds, which are localized in direct apposition to the presynaptic active zones containing synaptic vesicle clusters and membrane proteins that allow for efficient neurotransmitter release [71, 54], the molecular signals controlling the architecture of functional mature NMJs have not been fully elucidated.

Neurotrophins (NTs) are a family of growth factors that play a wide variety of functions in the nervous system through their binding to and activation of specific tyrosine kinase receptors (Trks) [10]. Effects on neuronal survival and neuronal growth are mainly triggered by binding of the nerve growth factor (NGF) to TrkA, the brain-derived neurotrophic factor (BDNF) and NT-4 to TrkB, and the NT-3 to TrkC [11, 33]. In addition, all NTs and their non-processed forms (pro-NTs) bind to the pan-NT receptor p75 (p75NTR), a multifunctional signaling receptor that belongs to the tumor necrosis factor receptor family [10]. Inhibition of axonal pruning, long-term depression and developmental or injury-induced apoptosis mainly rely on the binding of NTs and pro-NTs to p75NTR [22, 85, 93].

While p75NTR is widely expressed in different neuronal and glial populations in the develo** nervous system, its expression is down-regulated towards adulthood [57, 95]. All three NMJ cellular components retain low p75NTR expression levels at adult stages [25,26,27]. Even though p75NTR null mice (p75NTR−/−) display delayed NMJ synaptic refinement during early post-natal development, these phenotypes become soon restored [37]. Remarkably, p75NTR is strongly re-expressed in motor neurons and Schwann cells in conditions that negatively affect the nervous system [35]. These include experimental paradigms of nerve injury [39, 65, 79, 89, 91] and of amyotrophic lateral sclerosis [41, 52, 72], a neurodegenerative disease characterized by NMJ disruption and subsequent motor neuron death [59]. Cumulative evidence has demonstrated that p75NTR up-regulation impairs nervous system repair [2, 20, 35, 79, 88] and, consistently, p75NTR targeting has emerged as a therapeutic alternative to delay damage or disease progression [56, 54], we quantified postsynaptic structures containing a single (i.e. still maturing) or multiple peripheral openings (i.e. fully mature) (Fig. 1d). NMJs from 2-month-old p75NTR−/− mice show a significant decrease in the proportion of mature pretzel-like shapes, compared to WT controls (Fig. 1e). In 3D projections of NMJs from both genotypes (Additional file 3: Figure S3), we quantified significantly decreased values of postsynaptic area (Fig. 1f), surface (Fig. 1g) and volume (Fig. 1h), consistent with the relative abundance of less complex maturing structures found in p75NTR−/− mice. Based on these findings, we next analyzed potential postsynaptic NMJ defects at the ultrastructural level (Fig. 1i). Whereas the secondary clefts of control NMJs are long, thin, and closely spaced, these structures are reduced in number (Fig. 1j) and give rise to decreased postsynaptic perimeter in p75NTR−/− mice (Fig. 1k). Our quantitative analyses revealed that other parameters, such as the depth and width of the secondary folds, as well as the width of the primary fold, are not significantly affected in p75NTR−/− mice (Fig. 1l–n). Together, our findings reveal that the absence of p75NTR negatively affects the organization of the NMJ postsynaptic domain.

Fig. 1
figure 1

Abnormal NMJ postsynaptic complexity and structure in p75NTR−/− mice. a-c Whole-mounts of LAL muscles of 2–4 months old WT and p75NTR−/− mice were stained to reveal presynaptic motor terminals (2H3 plus SV2 antibodies, red), postsynaptic AChRs (BTX, green) and terminal Schwann cells (S-100 antibody, white). Laser confocal images of the R3 innervation region of WT and p75NTR−/− mice LAL muscle (a) show full coverage of AChR pretzels by presynaptic motor axons and terminal Schwann cells (b). Scale bar: 50 μm. c Quantification of the apposition of postsynaptic AChR pretzels by presynaptic motor axons. The plots correspond to > 35 NMJs per mice. d-h Diaphragm muscles from 2 months old WT and p75NTR−/− mice were stained with fluorescent-conjugated BTX to reveal AChR aggregates. d Postsynaptic apparatuses were classified into those having one or multiple peripheral openings and the relative abundance of these shapes was quantified (e). The plot corresponds to 130–220 NMJs per mouse. From 3D images, the area (f), surface (g) and volume (h) of individual pretzels were quantified. The plots correspond to 50–100 NMJs per animal. i–n Diaphragm NMJs from 2 to 4 months old p75NTR−/− and WT mice were analyzed by transmission electron microscopy. i Representative images of WT and p75NTR−/− NMJs. Scale bar: 500 nm. j The number of secondary folds is also expressed regarding the pre- and postsynaptic apposition length. k The postsynaptic perimeter was quantified as the length of the muscle membrane within the secondary folds (red line in i) expressed regarding the apposition length between the pre- and postsynaptic contact zone. l The depth of secondary folds was measured in a straight line traced between the beginnings of the invagination to the deepest point of the fold (green dotted line in i). m Quantification of the width of the secondary fold (blue line in i). n Quantification of the primary fold width, corresponding to the synaptic cleft width (black line in i). The plots correspond to 3–4 NMJs per animal. The bars represent the mean ± SEM of n = 3 (WT and p75NTR−/−) mice in (a–c) and (i–n), n = 8 (WT and p75NTR−/−) mice in (d–h). n.s, non-significant, *p < 0.5, **p < 0.01, ***p < 0.001, two-way ANOVA (e), or unpaired t-test (c, f–h, j–n)

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p75NTR null mice muscles display increased fatigue, decreased force production and histological alterations

We next aimed to correlate our morphological observations with functional NMJ parameters by analyzing the effect of sciatic nerve stimulation on hind limb muscle force generation and fatigue. After single twitch stimulation (Fig. 2a) we found no differences in the latency period (Fig. 2b) and a slight but significant decrease in the contraction time in p75NTR−/− mice (Fig. 2c). After repetitive nerve stimulation protocols (Fig. 2d), we found that p75NTR−/− mice muscles displayed a significantly increased fatigue after 30s of tetanic 100 Hz nerve stimulation (Fig. 2e). We complemented these studies with electromyography recording after presynaptic stimulation (Fig. 2f). We found that p75NTR−/− mice display decreased CMAP, a feature that was enhanced after repetitive nerve stimulation (Fig. 2g). After 7 s of repetitive nerve stimulation, single CMAP values were similar in control and p75NTR−/− mice muscles (Fig. 2g). Together, these results reveal that p75NTR−/− mice display altered nerve-induced muscle responses.

Fig. 2
figure 2

The p75NTR−/− mice display NMJ transmission alterations and accelerated nerve-dependent muscle fatigue. Time course of the mean force measurements (as a percentage of maximal force) for hind limb muscles after sciatic nerve stimulation of 2–4 months old WT and p75NTR−/− mice. First, the phases of contractile response elicited by 1 Hz sciatic nerve stimulation were measured. a Representative traces of 1 Hz stimulation protocol. No changes were observed in the latency period (b) of single-twitch stimulations, whereas a slight but significant decrease was observed in the contraction time (c) in p75NTR−/− muscles compared to WT controls. Second, force decline was determined after incomplete (10 Hz) and complete (100 Hz) tetanus. d Representative traces of 10 and 100 Hz stimulation protocols. A significant acceleration of muscle fatigue was observed at 30s of 100 Hz stimulation in p75NTR−/− compared to WT mice (e). f Representative traces of electromyographic recording after repetitive nerve stimulation. g Quantification of CMAP shows that p75NTR−/− mice display decreased impaired neuromuscular activity after the first and after repetitive presynaptic stimulation (RNS). The results represent the mean ± SEM of n = 3–4 (WT), n = 4–7 (p75NTR−/−) in (a–e) and n = 7 (WT), n = 8 (p75NTR−/−) in (f–g). n.s., non-significant, *p < 0.5, ***p < 0.001, unpaired t-test (b–c, g), or two-way ANOVA (e)

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To analyze if sustained defective synaptic activity resulted in skeletal muscle defects in p75NTR−/− mice, we first quantified the total levels of the sarcomeric proteins myosin heavy chain (MyHC) and troponin C (TnC) by Western blot (Fig. 3a). Compared to the loading control GAPDH, we found no differences in the levels of both proteins in the different genotypes (Fig. 3b). Next, we measured the force elicited after direct stimulation of the TA muscle at different frequencies. Our findings show a significant decrease in the net isometric force in the range of 30 to 100 Hz stimulation in p75NTR−/− TA muscles (Fig. 3c). These findings correlate with a significant reduction in the percentage of maximum isometric contraction force developed by p75NTR−/− mice muscles after 60 Hz stimulation (Fig. 3d). As these findings reveal defective muscle contraction in p75NTR−/− mice, we next studied muscle structure by histological analyses of transversal TA muscle cryosections (Fig. 3e). Our results show no gross differences in muscle fiber distribution or mononuclear infiltration (H&C staining). We also observed no differences in spontaneous muscle fiber degeneration/regeneration cycles, as cryosections from both genotypes show similar WGA/DAPI staining (Fig. 3e), a similarly low proportion of myofibers displaying central nuclei (Fig. 3f), and the absence of fibers expressing an embryonic form of MyHC (Additional file 2: Figure S2b), a molecular marker of muscle regeneration [32]. To evaluate potential differences in inter-fiber extracellular matrix deposition, we first performed immunohistochemical staining (Fig. 3g) and Western blot analyses (Fig. 3h, i) to study the levels of fibronectin. We found no differences in fibronectin levels or distribution in TA muscle samples from both genotypes. To complement these studies, we standardized the second harmonic generation technique, a two-photon laser confocal analysis that allows label-free imaging of collagen fibers at high resolution on native tissue [23]. Following this approach, we consistently found that the levels of collagen deposition, as well as the normally linear organization of collagen fibers, are not affected in skeletal muscles of the p75NTR−/− mice, compared to controls (Fig. 3g). We next evaluated muscle fiber plasticity by histochemical staining to reveal NADH-thioreductase (NADH-TR) activity (Fig. 3j). Quantification shows no differences in the relative composition of p75NTR−/− and control TA muscles regarding fast (light blue), intermediate (blue), and slow-twitch (dark blue) fibers (Fig. 3k). However, we found a significant reduction in the cross-sectional area (CSA) of muscle fibers of p75NTR−/− mice, evidenced by a strong increase in the proportion of low caliber fibers and a corresponding decrease in higher caliber fibers of both, slow and fast-twitch muscle fibers (Fig. 3l–m). To analyze if the decrease in muscle fiber size observed in p75NTR−/− mice was due to atrophy, we conducted Western blot experiments to analyze the levels of MuRF-1 and atrogin-1, two E3 ubiquitin ligases that act as key regulators of ubiquitin-mediated protein degradation in skeletal muscle [4]. We detected similar low levels of both proteins in TA muscle samples from both, control and p75NTR−/− mice (Additional file 2: Figure S2c). Together, these findings evidence that the absence of p75NTR results in functional NMJ defects, which correlate with altered structure and contractile capabilities of skeletal muscles.

Fig. 3
figure 3

Skeletal muscle properties of p75NTR−/− mice. a Total protein samples of Gastrocnemius muscles from 2 to 4 months old WT and p75NTR−/− (n = 4) mice were analyzed by Western blot using specific antibodies to detect myosin heavy chain (MyHC) and Troponin C (TnC). The levels of GAPDH were used as loading control. b Quantification of the relative levels of the sarcomeric proteins was performed by band intensity densitometry and expressed as a ratio of GAPDH band intensity. c Contraction force curve as a function of stimulation frequency (Hz) in isolated TA muscle. d Maximal isometric force was determined in WT (n = 6) and p75NTR−/− (n = 4) mice. e Cross-sections of TA muscles from 2-months-old WT and p75−/− mice were evaluated by hematoxylin/chromotrope staining (H&C) and WGA plus DAPI staining. Bar: 50 μm. f The percentage of myofibers bearing central nuclei was quantified in WGA/DAPI-stained cryosections of TA muscles from WT (n = 8) and p75NTR−/− (n = 8) mice. g Cross-sections of TA muscles from both genotypes were stained to analyze inter-fiber protein deposition. Bright field images (fist column) display DAPI nuclear (blue) plus BTX NMJ (magenta) staining. The same sections were processed for immunohistochemical detection of fibronectin (second column), as well as for the two-photon laser-based second harmonic generation technique (third column, SHG) to detect collagen fibers. The insets show high magnification images at the perimysium. The fourth column show the merged images. Bar: 50 μm. h Total protein samples of TA muscles from WT and p75NTR−/− (n = 3) mice were analyzed by Western blot using a specific antibody to detect fibronectin. The levels of β-actin were used as loading control. i Quantification of the relative levels of fibronectin was performed by band intensity densitometry and expressed as a ratio of β-actin band intensity. j Cross-sections of TA muscles from both genotypes were histochemically stained to detect NADH-TR activity. Scale bar: 50 μm. k Quantification of fiber types in at least 400 fibers per WT or p75NTR−/− mice. Results are expressed as a percentage of the total quantified fibers in a central region of interest. l Distribution histogram of the cross-sectional area (CSA) of slow-twitch fibers. m Distribution histogram of the CSA of fast-twitch fibers. The results represent the mean ± SEM of n = 8 (WT and p75NTR−/−) mice by quantifying at least 100 fibers per mice. n.s., non-significant, *p < 0.5; **p < 0.1; ***p < 0.01, unpaired t-test (b, d, f, i) or two-way ANOVA (c, k–m)

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p75NTR null mice display abnormal motor behavior

To study if the alterations in muscle contraction, ultrastructure, and function of the neuromuscular synapse found in p75NTR−/− mice resulted in defective motor behavior, several motor coordination and balance tests were performed. We first found that p75NTR−/− mice display altered walking gait, evidenced by an increase in the step distance (Fig. 4a) and in the traces between fore and hind limbs (Fig. 4b). Also, p75NTR−/− mice displayed a shorter latency time to fall in the rotarod test (Fig. 4c), and required more time to reach the end, or fell out, either in the triple horizontal bars (Fig. 4d) or in the static bar tests (Fig. 4e). As motor alterations are due to anatomical defects in mice models displaying NMJ phenotypes [58], we also measured the kyphotic index (i.e. abnormal spine curvature); however, no differences were found between p75NTR−/− and WT mice (Fig. 4f–g). Based on our previous findings, we next evaluated whether the absence of p75NTR results in force impairment in the context of the entire animal. Our results show that p75NTR−/− mice are able to hold their body weights in the inverted grid test, as WT mice do (Fig. 4h). However, when challenged to hold increasing weights with their fore limbs, p75NTR−/− mice show a significantly impaired performance (Fig. 4i). Thus, the absence of p75NTR results in defective locomotor behavior.

Fig. 4
figure 4

Altered motor performance and muscle strength in p75NTR−/− mice. a–e Young (2–4 months old) WT (n = 8) and p75NTR−/− (n = 8) mice were challenged in different motor tests. In the footprint test, p75NTR−/− mice displayed increased distance between steps (a) and increased distance between the fore and hind limbs steps (b) compared to WT controls. In the accelerating rotarod test, the latency time in two consecutive days was significantly decreased in p75NTR−/− mice compared to WT controls (c). Similarly, in the triple horizontal bar test (d) and the static bar test (e) p75NTR−/− mice had reduced scores and latency, respectively, compared to WT mice. f Representative X-ray images of WT and p75NTR−/− mice. The kyphotic index (KI) was calculated as the ratio between distances D1 and D2. g KI quantification show no differences between WT (n = 4) and p75NTR−/− (n = 3) mice. h–i Young (2–4 months old) WT and p75NTR−/− mice were challenged in two muscle strength tests. h In the Kondziela’s inverted screen test, no differences were detected between WT and p75NTR−/− mice. However, in the weights test (i), p75NTR−/− mice (n = 6) obtained a significantly impaired score compared to WT mice (n = 5). The results represent the mean ± SEM. n.s., non-significant, *p < 0.5; **p < 0.1; ***p < 0.01, unpaired t-test (a, b, g–i), or two-way ANOVA (c–e)

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The absence of p75NTR affects the ultrastructure of motor nerve terminals

Mature NMJs express low levels of p75NTR in motor axon terminals, terminal Schwann cells, and muscle fibers, in close vicinity to AChR aggregates [26, 27]. In order to gain insights on the cellular source of the p75NTR fraction potentially involved in postsynaptic NMJ organization and ultrastructural assembly, we first analyzed the aneural formation of complex AChR structures on cultured myotubes obtained from satellite cells of WT and p75NTR−/− mice (Fig. 5a). Our analyses revealed similar myotube formation and size in both genotypes (Fig. 5b). When seeded onto polyornithine/laminin matrices, myotubes assemble complex postsynaptic structures resembling those observed in vivo [45]. We categorized AChR aggregates into “immature plaques”, having homogenous AChR distribution, and “complex maturing forms”, corresponding to those having AChR-poor subregions forming “O-”, “C-” or “pretzel-like” shapes (Fig. 5c). Our morphological classification indicates no differences in the complexity of AChR aggregates amongst myotubes derived from p75NTR−/− and WT mice (Fig. 5d). Consistently, no differences were detected in the size of postsynaptic structures (Fig. 5e) or in the ability of myotubes from both genotypes to assemble complex postsynaptic structures, quantified as the number of AChR complex structures per myotube area (Fig. 5f). These findings suggest that the absence of the muscle-derived p75NTR receptor does not alter the assembly of mature postsynaptic structures in the p75NTR−/− mice in vivo.

Fig. 5
figure 5

Muscle-derived p75NTR is not required for nerve-independent AChR aggregation. Myoblasts from the gastrocnemius muscle of 2–4 months old WT and p75NTR−/− mice were induced to fuse into myotubes for 5 days onto poly-ornithine/laminin coated dishes. a Representative laser confocal images show myotubes from both genotypes stained to reveal actin (phalloidin, red) and AChR aggregates (BTX, green) Scale bar: 50 μm. b Quantification of myotube area per field show no differences amongst both genotypes. c Morphology of aneurally-induced complex AChR aggregates, which were classified into “plaques” and “complex shapes”, the latter including O-, C-, and pretzel-like shapes. d The relative abundance of AChR shapes was quantified and expressed as a fraction of the total AChR structures. e The area of AChR shapes was also determined. f Quantification of the number of AChR shapes expressed regarding the myotube area. The plots represent the mean ± SEM of n = 4 (WT and p75NTR−/−) mice. Three cultures for each animal and 30–50 AChR aggregates were analyzed per experiment. g–h For the in vivo two-color BTX method, LAL muscles from WT and p75NTR−/− mice were labeled with BTX-1 and the right hemi-LAL muscle was subsequently denervated by facial nerve transection. After a 7-day recovery period, LAL muscles were dissected and labeled with BTX-2. NMJs in maximum intensity projection images of LAL whole mounts (g; Bar: 50 μm) were categorized in “stable” or “dynamic” according to both BTX relative intensities, and quantified (h). n.s., non-significant, ***p < 0.01, unpaired t-test (b, e, f), or two-way ANOVA (d, h)

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As a way to investigate the possibility that muscle-derived p75NTR affects postsynaptic maintenance, the dynamics of AChRs after NMJ denervation were analyzed following an in vivo two-color BTX method [29]. With this aim, NMJ postsynaptic domains of LAL muscles were labeled with a non-saturating dose of a fluorescently tagged BTX (BTX-1) in live mice from both phenotypes and the right hemi-LAL muscle was subsequently subjected to denervation through facial nerve resection. After a 7-day recovery period, the pre-labeled muscles were dissected and labeled with a different fluorescently tagged variant of BTX (BTX-2). Using confocal microscopy, AChR aggregates were categorized in “stable” if BTX-1 and BTX-2 labels were similarly intense, or as “dynamic” if BTX-1 labelling was mostly absent and BTX-2 intensity was comparatively higher (Fig. 5g). As expected, denervation resulted in a significant increase in the proportion of dynamic AChR aggregates in LAL muscles from both, control and p75NTR−/− mice; however, no differences were detected in the proportion of dynamic AChR aggregates amongst denervated LAL muscles from p75NTR−/− and WT mice (Fig. 5h). These findings suggest that the absence of the muscle-derived p75NTR does not differentially affect the stability of mature postsynaptic structures after denervation.

Even though we did not find differences in the profile of muscle fiber innervation (Fig. 1a–c), our previous findings showing functional NMJ defects in p75NTR−/− mice prompted us to examine the presynaptic ultrastructure (Fig. 6a). Remarkably, quantitative analyses showed a drastic decrease in the total vesicle density in p75NTR−/− motor terminals (Fig. 6b). p75NTR−/− mice also displayed a significant reduction in the density of the readily releasable pool of vesicles (RRP) (Fig. 6c), as well as a strong decrease in the number of active zones per unit length of terminal apposition (Fig. 6d). No differences were found in the diameter of individual synaptic vesicles (Fig. 6e).

Fig. 6
figure 6

Reduced presynaptic vesicles in p75NTR−/− mice contribute to impaired synaptic transmission and muscle strength. a–e Diaphragm NMJs from 2 to 4 months old WT and p75NTR−/− (n = 3) mice were analyzed by transmission electron microscopy. a Representative images of WT and p75NTR−/− NMJs. Scale bar: 500 nm. b The total number of vesicles in each axon terminal was quantified as a fraction of the synaptic terminal area. c To quantify the RRP of vesicles, the number of vesicles within a 480 nm strip directly across active zones was counted. d Active zones (AZ) were quantified as electro dense regions located in direct apposition to postsynaptic secondary folds. The results are expressed as a function of the length of pre/postsynaptic apposition. e Vesicle diameter was measured in 40–110 vesicles per nerve terminal (n = 3–4 NMJs per mice). f–g Pharmacological inhibition of acetylcholinesterase with daily subcutaneous administration of pyridostigmine bromide partially rescued muscle strength (n = 5) (f) and NMJ transmission (n = 4) (g) of p75NTR−/− mice. The results represent the mean ± SEM. n.s., non-significant, *p < 0.5; **p < 0.01, unpaired t-test. b–d, f, g, or two-way ANOVA (e)

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Considering that p75NTR−/− mice display decreased number of vesicles in the motor axon terminal as well as postsynaptic morphological defects, we hypothesized that impaired neurotransmission could contribute to deficient NMJ function, subsequent muscle contraction impairment, and defective locomotor performance. To experimentally address this idea, pharmacological interventions were conducted in which the enzyme acetylcholinesterase was inhibited by subcutaneous administration of pyridostigmine bromide. After daily treatment with the drug, p75NTR−/− mice were challenged in the weights test to evaluate muscle strength. Our results show that p75NTR−/− mice had partially rescued strength at 2 and 3 days after daily drug administration compared to p75NTR−/− mice treated with physiological saline (Fig. 6f). Acetylcholinesterase inhibition also improved the electromyographic recording of p75NTR−/− NMJs, reflected in increased CMAP activity of p75NTR−/− pyridostigmine treated animals from 1 to 3 days (Fig. 6g). Our results also showed that the positive effects of acetylcholinesterase inhibition are not sustained in time, as CMAP values did not increase after 5 days of treatment (Fig. 6g). Altogether, these findings reinforce the idea that impaired neurotransmission contributes to deficient NMJ function in p75NTR−/− mice.

NMJs of p75NTR null mice display unaltered distribution of BDNF and TrkB

It has been demonstrated that the levels of NTs and their Trk receptors are altered in conditions affecting the neuromuscular synapse. For instance, hind limb muscles of amyotrophic lateral sclerosis mouse models display a significant decline in the number of NMJs expressing BDNF, NT-4, GDNF, p75NTR, TrkB, and TrkC [31]. Therefore, as a first hint to analyze the potential involvement of NT-dependent signaling on the morphological and functional alterations observed in p75NTR−/− mice, we analyzed the levels of expression of BDNF and its TrkB receptor at the NMJ, as BDNF/TrkB signaling plays important roles on NMJ organization, including synaptic vesicle availability [34, 75]. With that aim, cryosections of TA muscles from control and p75NTR−/− mice were subjected to immunohistochemistry to detect BDNF and an active form of TrkB (p-TrkB Y816) (Fig. 7). Our findings show that NMJs from control and p75NTR−/− mice display positive staining for BDNF and p-TrkB Y816 (Fig. 7a). Immunofluorescence quantification shows that the vast majority of NMJs express both proteins in TA muscle cryosections of both genotypes (Fig. 7b, c). Following Western blot experiments, we analyzed the levels of TrkB and BDNF. We also included the antibody that specifically detects active TrkB when phosphorylated at residue Y816 of both, the glycosylated (145 kDa) and the unprocessed (110 kDa) forms of TrkB [38]. Our results show that the total levels of BDNF and TrkB are not significantly altered in p75NTR−/− mice muscles (Fig. 7d). Consistently, immunoblot quantifications show that the levels of pro-BDNF (the precursor form of BDNF) or the active p-TrkB Y816 form of TrkB were unchanged by the absence of p75NTR at the NMJ (Fig. 7e, f). Similarly, the levels and distribution of BDNF and TrkB in samples of sciatic nerve and the spinal cord of p75NTR−/− mice were similar to control mice (Additional file 4: Figure S4). Together, these findings suggest that BDNF/TrkB signaling is not significantly altered at the neuromuscular synapse of p75NTR−/− null mice.

Fig. 7
figure 7

Unaltered BDNF/TrkB signaling at the NMJ of p75NTR−/− mice. a TA muscle cryosections from WT and p75NTR−/− mice were double-labeled with antibodies (green) to detect p-TrkB Y816 or BDNF, along with BTX (red). b–c Quantification of the percentage of NMJs labeled with BNDF (b) or with p-TrkB Y816 (c). d Total protein samples of TA muscles from WT and p75NTR−/− (n = 3) mice were analyzed by Western blot using specific antibodies to detect p-TrkB Y816, total TrkB, and BDNF. The levels of GAPDH were used as loading control. e–f BDNF/TrkB signaling was estimated by band intensity densitometry of BDNF (e) and by the Y816 phosphorylation of both, the glycosylated (145 kDa) and the unprocessed (110 kDa) forms of TrkB and expressed as a ratio of the corresponding bands detected with an anti TrkB antibody (f). n.s., non-significant, unpaired t-test

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Discussion

Dysfunctions of the NMJ are caused by traumatic spinal cord or peripheral nerve injuries as well as by severe motor pathologies [35, 36, 59]. Despite the remarkable regenerating ability of the peripheral nervous system, delayed NMJ regeneration paradigms show that, even though muscles are reached by motor axons and re-build morphologically normal NMJs after long-term denervation, regeneration after a critical period is not associated with a positive functional outcome in distal muscles [53, 70], suggesting synaptic rather than regenerative failures after critical periods of time. In mice models of amyotrophic lateral sclerosis, it has been demonstrated that NMJ disruption precedes subsequent motor neuron death [59], demonstrating a critical primary role for NMJ maintenance in the etiology of this neurodegenerative disease. Together, these findings reveal that cells at the damaged NMJ niche express signals that impair synaptic maintenance and repair [78]. Cumulative evidence shows that p75NTR is likely one such molecule. Although Schwann cell-derived p75NTR plays a major role on the perinatal elimination of muscle fiber poly-innervation [37, 92], NMJs of P14 p75NTR−/− mice showed no evident alterations regarding AChR clustering and their innervation profile [37], suggesting a minor function for this receptor on the maintenance and function of mature NMJs. Remarkably, various experimental paradigms of nerve injury result in strongly increased p75NTR expression in motor neurons [19, 42, 69] and Schwann cells [30, 65, 79]. Also, p75NTR expression is up-regulated in spinal cord motor neurons in mice models of amyotrophic lateral sclerosis [41, 52, 72]. Rather than a positive outcome given by its role as a receptor for NTs, cumulative evidence has demonstrated that p75NTR up-regulation impairs nervous system repair, a feature related to its additional role as a cell death mediator in various neuronal and glial populations [1, 8, 20,21,22]. Indeed, p75NTR inhibition has been successfully tested as a pharmacological target to delay disease progression [35, 54]. Our results also show a significant alteration in motor coordination and balance in p75NTR−/− mice, as previously reported [64, 68, 96]. In this context, mice null for p75NTR in the cerebellar external granular layer (EGL) also display altered motor performance [96]. Additionally, it has been shown that the absence of p75NTR in hippocampal neurons reduces neurogenesis, resulting in some behavioral alterations but, interestingly, not in locomotor defects [9, 12]. Our studies provide novel evidence showing that, in addition to its effects on the central motor coordination, the absence of p75NTR specifically alters NMJ pre- and postsynaptic organization, a feature that can at least partially explain the observed defects in muscle structure and locomotor performance.

The mouse model used throughout our studies express a p75NTR isoform lacking cysteine repeats 2, 3, and 4 and express a truncated form of p75NTR receptor that could potentially contribute to the phenotypes that we and others have described in these mice [86]. However, polypeptide fragments derived from this truncated form of p75NTR are expressed at low levels, as they are susceptible to degradation by the α- and γ-secretases, as well as by the proteasome system; indeed, Trk-dependent signaling activates the α-secretase processing and endosomal targeting of these p75NTR-derived fragments [83]. If any, the p75NTR protein fragments expressed by p75NTR exon III null mice could play a beneficial effect in the context of the NMJ, as they have been shown to increase BDNF-TrkB dependent survival of motor neurons in vitro and in vivo in the hSODG93A mice model of amyotrophic lateral sclerosis [56]. Therefore, future experiments using complete p75NTR null models will complement our observations regarding the critical effects that p75NTR inhibition exerts in the organization of mature NMJs.

Our functional studies showed that repetitive presynaptic stimulation resulted in increased muscle fatigability, as well as in impaired muscle force generation and CMAP values in p75NTR−/− mice. Interestingly, we observed a rescue of a single CMAP value in p75NTR−/− mice 7 s after repetitive nerve stimulation. A comparative protocol is commonly used in the clinic to discriminate the pre or postsynaptic etiology of myasthenic diseases; CMAP values at rest are reduced but display a strong rescue 10 s after maximal voluntary contraction (MVC) in myasthenic diseases of presynaptic origin (such as the Lambert-Eaton myasthenic syndrome) due to post-activation facilitation (PAF) [49]. Also, while acetylcholinesterase inhibition increased CMAP values at days 1 and 3, this effect was lost at day 5, a finding consistent with the observation that sustained administration of cholinesterase inhibitors as a monotherapy are minimal and not sustained in time in presynaptic myasthenic diseases [84]. Therefore, despite the limitations of our approach to be compared to the clinical practice, and although a postsynaptic effect cannot be discarded, we believe that our electromyographic recordings, along with the transmission electron microscopy studies, strongly suggest a main presynaptic defect at the NMJ of p75NTR−/− mice. We speculate that this sustained upstream presynaptic defect impairs neurotransmitter availability, which is manifested in downstream defects in NMJ morphology, muscle fiber structure, and locomotor performance.

p75NTR is a multifaceted receptor with the ability to signal through NT-dependent and independent pathways. Even though our results suggest that BDNF/TrkB signaling is not significantly affected at the NMJ of p75NTR−/− mice, several lines of evidence relate NT signaling with the defects we found in these mice. For instance, acute antibody-dependent blocking of p75NTR impairs ACh release in immature and mature neuromuscular synapses via a BDNF/TrkB-dependent mechanisms that regulates the phosphorylation of presynaptic proteins [24, 27, 62, 75]. Our findings showing that the availability of synaptic vesicles is severely compromised in the absence of p75NTR are also related to NT signaling. Indeed, decreased expression of integral synaptic vesicle proteins has been reported in cultures of neurons derived from conditions with altered NT-dependent signaling, such as TrkB, TrkC and BDNF deficient mice [18, 55, 67, 80]. In addition, NTs have been reported to be involved in synaptic vesicle fusion to the plasma membrane in nerve terminals of central synapses [55, 67, 80, 82]. In frog nerve-muscle co-cultures, the expression of synapsin I, a presynaptic protein that distributes in myotube-contacting neurites, is increased by NT3 [87]. Interestingly, similar to our findings in the p75NTR null mice, NT3+/− mice exhibit impaired muscle contraction force and lower synaptic vesicle recycling in motor axon terminals [73]. We also found that p75NTR−/− mice displayed increased susceptibility to fatigue after tetanic nerve stimulation, a phenotype similar to that observed in NT4−/− mice [3]. The idea that Trk receptors and p75NTR exert comparable effects at the NMJ is also supported by the functional consequences of reducing TrkB and p75NTR. Indeed, heterozygous TrkB+/− mice show decreased contractile force and muscle fiber CSA [44], as we found in p75NTR−/− mice. In turn, activation of TrkB signaling in mice null for TrkB.t1, an endogenous truncated dominant-negative variant of the receptor, results in increased isometric contraction force and increased CSA [17], exactly opposite to what we found in p75NTR−/− mice. An interesting comparison also emerges regarding TrkB and p75NTR inhibition in a pathological context. Indeed, inhibition of TrkB expression in motor neurons of an amyotrophic lateral sclerosis mice model results in beneficial delaying effects on disease progression [97], whereas TrkB deletion impairs NMJ structure and function [28, 44]. Together with our findings, these results reveal the need for future research to elucidate how Trk and p75NTR receptors act at the mature NMJ and how the signaling pathways controlled by these receptors are balanced to contribute to the correct apposition between the nerve terminal and the post-synaptic muscle domain. Our findings also contribute to a more comprehensive view of the effects that therapeutic attempts to target p75NTR may have at the neuromuscular connectivity.

Conclusion

We conclude that p75NTR plays essential roles in neurotransmitter availability and on the organization of mature NMJs. Our results in p75NTR exon III null mice describing uncharacterized phenotypes at the levels of synaptic function, muscle fiber structure, force production, and locomotor performance should be considered in the development of therapeutic strategies focused on targeting p75NTR for NMJ repair.