Molecular Neurobiology https://doi.org/10.1007/s12035-018-1021-0
HERC1 Ubiquitin Ligase Is Required for Normal Axonal Myelination in the Peripheral Nervous System Sara Bachiller 1 & María Angustias Roca-Ceballos 2 & Irene García-Domínguez 2 & Eva María Pérez-Villegas 1 & David Martos-Carmona 1 & Miguel Ángel Pérez-Castro 2 & Luis Miguel Real 3 & José Luis Rosa 4 & Lucía Tabares 5 & José Luis Venero 2 & José Ángel Armengol 1 & Ángel Manuel Carrión 1 & Rocío Ruiz 1,2 Received: 23 January 2018 / Accepted: 16 March 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract A missense mutation in HERC1 provokes loss of cerebellar Purkinje cells, tremor, and unstable gait in tambaleante (tbl) mice. Recently, we have shown that before cerebellar degeneration takes place, the tbl mouse suffers from a reduction in the number of vesicles available for release at the neuromuscular junction (NMJ). The aim of the present work was to study to which extent the alteration in HERC1 may affect other cells in the nervous system and how this may influence the motor dysfunction observed in these mice. The functional analysis showed a consistent delay in the propagation of the action potential in mutant mice in comparison with control littermates. Morphological analyses of glial cells in motor axons revealed signs of compact myelin damage as tomacula and local hypermyelination foci. Moreover, we observed an alteration in non-myelinated terminal Schwann cells at the level of the NMJ. Additionally, we found a significant increment of phosphorylated Akt-2 in the sciatic nerve. Based on these findings, we propose a molecular model that could explain how mutated HERC1 in tbl mice affects the myelination process in the peripheral nervous system. Finally, since the myelin abnormalities found in tbl mice are histological hallmarks of neuropathic periphery diseases, tbl mutant mice could be considered as a new mouse model for this type of diseases. Keywords Inherited peripheral neuropathies . Myelin . Proteasome . Charcot-Marie-tooth . Neuromuscular junction
Introduction The tambaleante (tbl) mutant mouse presents tremor, unstable gait, abnormal posture of the hind limbs, and loss of Purkinje Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12035-018-1021-0) contains supplementary material, which is available to authorized users. * Rocío Ruiz
[email protected] 1
Department of Physiology, Anatomy and Cellular Biology, University of Pablo de Olavide, Seville, Spain
2
Department of Biochemistry and Molecular Biology, School of Pharmacy, University of Seville, and Instituto de Biomedicina de Sevilla-Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Calle Profesor García González 2, 41012 Sevilla, Spain
3
Unit of Infectious Diseases and Microbiology, Valme University Hospital, Seville, Spain
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Departament de Ciències Fisiològiques II, IDIBELL, Campus de Bellvitge, Universitat de Barcelona, L’Hospitalet de Llobregat, E-08907 Barcelona, Spain
5
Department of Medical Physiology and Biophysics, School of Medicine, University of Seville, Seville, Spain
cells in the cerebellum [1–4]. Recently, we described an impairment in motor performance prior to the cerebellar neurodegeneration, consisting of a reduction of the motor end-plate area, a less efficient neuromuscular activity in vivo, and an impaired evoked neurotransmitter release at the neuromuscular junction (NMJ) [5]. Genetically, his complex phenotype is based on a single nucleotide transition within the Herc1 (HECT and RLD Domain Containing E3 Ubiquitin Protein Ligase Family Member 1) gene at position 1448. This change generates an overexpress-mutated protein with a Gly483Glu amino acid substitution that does not exert a dominant negative effect [1]. The HERC1 protein belongs to the ubiquitin–proteasome system (UPS) [6–8]. UPS plays a key role in the protein degradation pathway essential for neuronal homeostasis. It has been postulated that alterations in the UPS pathway are involved in the pathogenesis of several neurodegenerative disorders including neuromuscular diseases such as spinal muscular atrophy (SMA), spinal and bulbar muscular atrophy (SBMA), X-spinal muscular atrophy and X-linked juvenile and adult-onset amyotrophic lateral sclerosis (ALS) [9–13]. Likewise, the development of severe forms of peripheral
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neuropathies such as Charcot-Marie-Tooth disease (CMT) are linked to alteration of this pathway [14–16]. Neuromuscular diseases are grouped into more than 150 different types and are progressive in nature, most of them has a genetic origin (and hence inheritable) and its main feature is the loss of muscle strength. There is growing evidence that glial cells have a major impact on neurodegenerative processes [17]. Indeed, glial cells collaborate in the functionality of neurons in both the central and peripheral nervous systems [18, 19]. Recent studies suggest that glial anomalies contribute in neuromuscular pathogenesis such as ALS and SMA [20, 21]. In fact, Survival Motor Neuron protein (SMN) restoration in Schwann cells improves the neuromuscular function and reverses myelination disorders observed in the SMA mouse model [22]. Interestingly, UPS has been found as one of the altered systems in the Schwann cells of SMA mice [13]. Taken together, these data formed the basis to investigate the glial cell component in the tbl mutant mouse using a broad range of ex vivo and in vitro approaches. In this work, it is firstly describe an anomalous location of somatic processes of terminal Schwann cells (tSCs) in tbl NMJs along with an alteration in axonal wrapping and myelin sheath thickness. These changes seem to be correlated with the dysregulation of the Akt signal pathway in the tbl mutant mouse.
Materials and Methods Animal Model The experimental mice were obtained by breeding pairs of tambaleante (tbl) mutation carriers. Control and mutant mice were genotyped by PCR as described previously [1]. The control mice used were age-matched littermates of the mutants (2–4 month of age [5]). All experiments were performed according to current Spanish legislation RD 53/2013 governing experimental animal care (BOE 08/02/2013).
Intracellular Recording and Analyses Mice were anesthetized with tribromethanol 2% and sacrificed by exsanguination. The Levator Auris Longus (LAL) muscle is a flat, thin, and superficial muscle and is easy to manage in this kind of analyses [23]. The muscle was dissected with its nerve branches intact and pinned to the bottom of a 2-ml chamber on a bed of cured silicone rubber (Sylgard, Dow Corning). These preparations were perfused continuously with the following solution (in mM): 125 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 25 NaHCO3, and 30 glucose. This solution was continuously gassed with 95% O2 and 5% CO2 to maintain its pH at 7.4. Recordings were obtained at room temperature (22–23 °C). Ex vivo electrical stimulation was
performed by means of a suction electrode, applying 10 stimuli at 100 Hz (0.2 ms square-wave pulses; 2–40 V amplitude). Evoked end-plate potentials (EPPs) were recorded and analyzed as described previously [24–27]. Muscle contractions were blocked with μ-conotoxin GIIIB (2–4 μM; Alomone Labs), a specific blocker of skeletal muscle voltage-gated sodium channels. Latency or delay, meaning the elapsed time between stimulation and postsynaptic response, was measured as the time from the positive peak of the artifact to the 10% onset of the postsynaptic event.
Immunofluorescence Whole-mount LAL muscles were incubated for 90 min in 4% paraformaldehyde and immunostained as described previously [5, 28]. Briefly, the muscles were bathed in 0.1 M glycine in PBS for 30 min, permeabilized with 1% (v/v) Triton X-100 in PBS for 1 h, and then incubated in 5% (w/v) BSA, 1% Triton X-100 in PBS for 1 h, to prevent unspecific staining. The tissue was then incubated overnight at 4 °C with the following primary antibodies against presynaptic and glial proteins: neurofilaments (NF: SC-51683; 1:500), synaptophysin (SYP: SC-9116; 1:500), and myelin basic protein (MBP: SC-13912; 1:500) from Santa Cruz Biotechnology and S100β (EP1576Y; 1:250) from GeneTex. The following day, the muscles were rinsed for 1 h in PBS containing 0.05% Triton X-100. After incubating for 1 h with the corresponding secondary antibodies (1:500; Alexa antibodies, Invitrogen) and 10 ng/mL rhodamine-Bungarotoxin (BTX; T0195, Sigma Aldrich) to label the postsynaptic terminal, as well as rinsing again with PBS containing 0.05% Triton X100 for 90 min, the muscles were mounted in 50% Glycerol for visualization. Fixed muscles were examined on an upright Leica DM 2500 confocal laser scanning microscope using a × 63 oil-immersion objective with a numerical aperture of 1.3. Images from control and mutant littermates were obtained at the same day and under equal conditions (laser intensities and photomultiplier voltages). A morphometric analysis of the fluorescently labeled structures was performed offline with Fiji ImageJ (W. Rasband, National Institutes of Health). Orthogonal views (Fig. 2A) consist in an automatic method from Image J to visualize 3D renderings using a Z-stack to display the XZ and YZ planes at a given point in the 3D image.
Western Blot Sciatic nerve proteins were obtained by means of TRIsure® method following the indications of the manufacturer (Bioline). Protein content of the samples was estimated according to the Bradford method using BSA standards with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) [29] and loading 30–60 μg of protein in each lane.
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Protein samples were separated by SDS-PAGE (10%) and transferred to a nitrocellulose membrane (Biorad). Membranes were exposed to blocking buffer (5% milk in Tris-Buffer Saline (TBST): 20 mM Tris-HCl, pH 7.5, 500 mM NaCl and 0.05% Tween-20) for 1 h at room temperature. Then, membranes were incubated overnight at 4 °C in blocking buffer (5% milk in Tris-Buffer Saline) containing the primary antibodies: anti-MBP (SC-13912; 1:500), anti-P-Akt (ser473) (SC-6985-R; 1:500), anti-Akt (SC-81434; 1:500), anti-P-Erk (SC-7383; 1:50), anti-Erk (SC-94; 1:500) from Santa Cruz Biotechnology, and anti-P-S6K1 (#9205; 1:1000) from Cell Signaling Technology. GAPDH antibody (Santa Cruz; SC-365062; 1:1000) was used as a loading control. After incubation with the primary antibodies, all membranes were washed in TBST and, incubated with peroxidaseconjugated anti-immunoglobulin secondary antibodies (DAKO, Produktionsvej, DK) at a dilution of 1:2500 for 1 h at room temperature in TBST. Proteins were visualized using western blotting chemiluminescence luminol reagent kit Western Clarity™ ECL Substrate (Bio-Rad). All experiments were repeated at least two times. The bands were analyzed by densitometry using the Fiji ImageJ gel analyzer (W. Rasband, National Institutes of Health). The bands were quantified as percent of GAPDH expression.
RT-PCR RT-PCR was performed as previously described [30]. Briefly, to measure the gene expression of Akt, mRNA was extracted from the sciatic nerve from both control and mutant mice using the RNeasy Mini kit from Qiagen. Using the Maxima™ First Strand cDNA Synthesis Kit for RT-PCR (Fermentas, Sweden), 1 μg mRNA was transformed into cDNA. Primers: gapdh: forward 5′ GTA GGC CAA GTT GCC TTG TCC GT 3′ and Reverse 5′ATG TTC CAG TAT GAC TCC ACT CAC G 3′; Akt: forward 5′-GAG AAG AGA CTC TGA GCA TC-3′ and reverse 5′-GTG CCA CTG AGA AGT TGT TG-3′.
Transmission Electron Microscopy The procedure followed here was previously reported [31]. Briefly, control and mutant mice were deeply anesthetized with tribromoethanol (2%, i.p.) and perfused transcardially with a mixture of 1% paraformaldehyde and 1% glutaraldehyde in 0.12 M phosphate buffer (PB, pH 7.2). Thereafter, the LAL muscle and the sciatic nerve were dissected out and immersed overnight in the same fixative at 4 °C. LAL longitudinal and transversal slices and 2-mm long blocks of the sciatic nerve were cut and incubated in 2% OsO4 in PB, stained in block with ethanolic 0.5% uranyl acetate, dehydrated with an increased gradient of ethanol, and embedded in Durcupan (Fluka®). Ultrathin sections (50–70 nm)
were obtained on a Leica EM UC7 ultramicrotome, collected on copper grids (150 and 300 mesh) and observed without counterstaining in a Zeiss Libra electron microscope at 80 kV (CITIUS, University of Seville). Quantitative analyses of the sciatic nerves of three controls and three tbl mice were conducted using the Fiji ImageJ [32]. Owing that Remak bundles vary along the longitudinal axis of the sciatic nerve [33], all observations were made on sections of the sciatic nerve proximal to their peroneal and tibial branches’ bifurcation. Ultrathin sections of the sciatic nerve were microphotographed at × 2000. Myelinated fibers, Remak bundles, and C-fibers were counted and measured in a 1200-μm2 surface area per mouse. The g-ratio was calculated by dividing d (mean axonal diameter) by D [mean fiber diameter (axon including myelin sheath)]. The axonal diameters were categorized in the following three groups: 0–3 μm, 3.01–6 μm, and > 6.01 μm, and abnormalities of NMSCs forming Remak bundles were in accordance with those described by Orita et al. [34].
Statistical Analysis The results were analyzed with the SPSS package release 24.0 for Windows (IBM SPSS Inc, Chicago, Illinois, USA), and unless otherwise stated, the data represent the mean ± SEM values. To compare quantitative variables between tbl and control samples, Student’s t test was used unless otherwise stated. A P value < 0.05 was considered statistically significant. BN^ (capital letter) refers to the number of mice, while Bn^ (lowercase) indicates the number of either NMJ or fibers. All data represent the analyses of at least three animals per genotype as previously performed [35].
Results Evoked Release Latency Is Increased in tbl Terminals The neurotransmitter release is altered in the NMJ of tbl mutant mice [5]. To elucidate if the conduction of the action potential is also affected in this mouse model, we measured the latency between the nerve stimulation and the evoked postsynaptic response. The tbl mice showed a higher latency than their littermate controls [control: 1.27 ± 0.12 ms, (n, N = 24, 3); tbl: 1.68 ± 0.08 ms, (n, N = 30, 4); P = 0.02] (Fig. 1A, B). The progressive lengthening of this latency during the stimulus train (delta delay) was observed in both mutant and control mice (Fig. 1A, C). However, the average latency showed an approximately twofold increase by the tenth EPP compared with control NMJs [Control: 0.14 ± 0.03 ms, (n, N = 24, 3); tbl: 0.26 ± 0.05 ms, (n, N = 30, 4); P = 0.04] (Fig. 1C).
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Fig. 1 Tambaleante (tbl) mutant mice show a progressive increment in synaptic delay. A Representative recordings of control (upper traces) and tbl (lower traces) EPPs during a 10-stimuli train at 100 Hz. The continuous gray line represents the first EPP in the train and the discontinuous line the last response. The black arrowhead points to
10% of the onset of EPP, and the gray one the stimulation artifact. B Mean delay from the first peak of the stimulation artifact to the initial 10% of the first evoked response in tbl and control. C Average delay increment as a function of the successive stimuli in tbl and control. *P < 0.05
Non-myelinating Schwann Cells Invade the Synaptic Cleft of the NMJ in tbl Mutant Mice
was normalized with respect to the BTX area, the level of intensity in mutant terminals was higher than controls [(Control: 0.096 ± 0.02, (n, N = 38, 3); tbl: 0.24 ± 0.03 a.u., (n, N = 39, 3); P = 0.02)] (Fig. 2E). The ultrastructural study in the LAL muscle (Fig. 2F–I) did not reveal substantial differences in the tbl presynaptic terminal structure at NMJs in comparison with control mice (i.e., synaptic vesicles were clustered at active zones on the presynaptic membrane) (Fig. 2F, H). Likewise, the junctional folds of tbl NMJs possessed the same morphological features as control NMJs (Fig. 2F–I, jf). Terminal Schwann Cells (tSCs) and their somatic processes covered the axon terminals (Fig. 2F, G, tSC). In control NMJs, tSCs somatic processes capped the presynaptic terminal without wrapping it, keeping the synaptic cleft between the pre- and postsynaptic areas free (Fig. 2H, yellow). However, some axon terminals of tbl NMJs were completely or partially isolated from the junctional folds by thin somatic processes of tSCs invading the synaptic cleft (Fig. 2I, yellow). This finding on the ultrastructural level could explain the differences in localization and intensity of tSCs’ immunolabeling observed in tbl NMJs and suggests that the tSCs interposition at the synaptic cleft interfered with preand postsynaptic interconnections. It could thus, at least in part, be responsible for the neurotransmission delay.
Possible reasons for the increment in EPP delay during the stimulation train could be the slowing in the conduction velocity of the axonal action potential and/or its passive propagation into the nonmyelinated regions of the pre-terminal [26, 36, 37]. To further investigate this hypothesis, we immunohistochemically studied the glial component of NMJ. The labeling of terminal Schwann cells (tSCs) with S100β antibody (a common marker of glial cells [38]) displayed a different localization of S100β immunoreactivity through NMJ of mutant mice compared with controls (Fig. 2A). Orthogonal view showed that Schwann cells (Fig. 2A, magenta) were interposed between presynaptic (Fig. 2A, green) and postsynaptic (Fig. 2A, red) terminals in tbl NMJ. The BTX area, as previously reported [5], was smaller in tbl NMJ than in controls (Fig. 2B) [Control: 309.92 ± 22.04 μm2, (n, N = 38, 3); tbl: 227.57 ± 16.77 μm2, (n, N = 39, 3); P = 0.041]. No differences in the S100β labeled area were observed between groups (P = 0.09) (Fig. 2C). Nonetheless, the quantification of the mean intensity in the tbl terminal showed a significant increase of S100β in comparison with controls [Control: 25.17 ± 4.1 a.u., (n, N = 38, 3); tbl: 46.23 ± 2.66 a.u, (n, N = 39, 3); P = 0.023] (Fig. 2D). This result could be explained by a possible increase in the permeability of the tbl muscles during the immunostaining protocol. However, when the mean intensity of the BTX labeling was compared, no difference between the two groups was found (data not shown; P = 0.84); therefore, this possibility can likely be discarded. In addition, when the mean of S100β intensity
Myelin Is Altered in tbl Nerve Terminals Myelin causes Schwann cells to wrap the motor axons until evolving to individual muscle fibers, which are then replaced by tSCs at the NMJ level [39]. Confocal laser microscopy images of LAL muscle showed immunolabeled Myelin
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Fig. 2 S100β location and mean intensity are altered in tbl mice. A Representative en face views of NMJs from the LAL muscle stained with Bungarotoxin-Rhodamine (BTX, red), anti-S100β (magenta) and anti-Neurofilaments (NF) + Sinaptophysin (SYP) (green). Images are Zstack projections and the scale bar 10 μm. Orthogonal view in control and mutant (right inset), the lower showing each merged image. B The mean postsynaptic areas were significantly smaller in mutant than in control terminals. C No difference was found in the mean of S100β labeled area between both groups. D The mean of S100β intensity was higher in tbl terminals in comparison with controls. The normalized mean intensity vs
BTX area was increased in tbl terminals in comparison with controls. F–I Electron microphotographs of the motor end-plate of levator auris longus muscle of control (F, H) and tbl (G, I) mice. In control mice, the terminal Schwann cells (tSC) somatic processes (F, H, arrowheads, yellow) cover the axon terminals (At) that appose directly to the sarcolemma junctional folds face (jf) separated through the synaptic cleft (sc). In contrast, some axon terminals of tbl NMJ are separated from the junctional folds (G, I; jf) by thin tSC somatic processes (G, I, arrowheads, yellow) that invade the synaptic cleft. Arrows point the active zones (az). Scale bars: 2 μm (F, G). *P < 0.05
Basic Protein (MBP) (green), co-localized with pre(magenta) and postsynaptic (red) areas only in the ~ 1% of control NMJs (Fig. 3A). However, the percentage of MBPSYP-BTX co-localization was ~ 11% in tbl NMJs [(Control: 1.08 ± 0.24%, (n, N = 13, 4); tbl: 11.43 ± 1.37%, (n, N = 14, 4); P = 0.005)] (Fig. 3A (white arrows), B). The qualitative increase of myelin at the end of motor nerves observed by immunolabeling was also accompanied by a quantitative increase in the amount of MBP protein measured by western blot in the sciatic nerve (Fig. 3C, D) (control: 83.75 ± 18.74%, (N = 6); tbl: 214.04 ± 63.33%, (N = 8); P = 0.04). To get insight into the possible myelin and/or axonal alteration in tbl mutant mice, we performed an ultrastructural study of the sciatic nerve. Signs of alteration and/or damage of myelin were clearly observed in the sciatic nerve of tbl mice. Non-compact myelin [40] was observed at the Schmidt-Lanterman incisures in tbl sciatic nerve
(Fig. 4B, C, arrows). At Ranvier nodes level (Fig. 5F and Supplementary Fig. 1), hypertrophic myelin foci that compressed the axon (Fig. 4C, F, G) were observed in mutant sciatic nerves. Furthermore, in transverse sections of the Ranvier nodes, the Schwann cells’ microvilli surrounding the nodal membrane [41] were substituted in tbl axons by irregular concentric myelin figures (Supplementary Fig. 1A & B). Myelin infoldings and thicker myelin sheaths with degenerative signs were also evident in the internode myelin in tbl sciatic nerves (Fig. 5). Thus, tomacula protruding inside the axon (Fig. 5B, arrowhead), misstructured myelin foci (Fig. 5I, K, L, asterisk) and invaginations of the myelin sheath (Fig. 5C, D, asterisks), whose growth compressed the axon were frequently observed in tbl sciatic nerves. The invaginations of myelin sheaths (Fig. 5B, F, arrows), appear in longitudinal sections as islands of myelin within
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Fig. 3 The MBP distribution is altered in tbl NMJ. A Representative en face views of NMJs from the LAL muscle stained with anti-MBP (green), BTX-Rho (red) and anti-SYP (magenta). Images are Z-stack projections and the scale bar 100 μm. Insets show an amplified image where triplecolocalization is evident (white arrows) in tbl LAL muscle. B Quantitative analysis showed an anomalous distribution of MBP
(white arrows in A) in tbl mutant mice at NMJs junctions’ level compare with controls. C Representative western blot for MBP and GAPDH in sciatic nerve proteins from control and tbl mice. D Results of MBP quantification in sciatic nerve. The results are expressed as % of intensity relative to GAPDH bands. N = 6 control and N = 8 tbl. *P < 0.05; **P < 0.005
the axon (Fig. 5J, arrow). To quantify these anomalies in myelin, great and medium-sized axons were randomly selected and the number of hypermyelination foci were calculated and expressed as the ratio of the number of axons (P < 0.0001; Fig. 5O). However, myelin alterations did not modify the compact myelin thickness (see Supplementary Fig. 1F–I). Furthermore, electron micrographs showed myelin invaginations entering within the axon in a finger-like fashion (Fig. 5M, arrowheads). The number of myelin lamellae did not vary but the intraperiod line was greater surrounding these invaginations (Fig. 5N). Finally, g-ratios were significantly decreased in mutant mice being more pronounced in smaller (control: 0.66 ± 0.01; tbl; 0.60 ± 0.01; P < 0.001) and medium-caliber axons (control: 0.68 ± 0.1; tbl: 0.61 ± 0.01; P < 0.001) (Fig. 5P, Q). In addition, we observed further disturbances in tbl myelinated axons, i.e., increased periaxonal space and anomalous inner mesaxon (see Supplementary Fig. 1C–E). Altogether, these results suggest that overexpression of mutated HERC1 protein leads to a profound myelin alteration.
Mutated Herc1 Produces Abnormalities in the Ensheathment of Axons by Non-myelinating Schwann Cells in Remak Bundles In normal peripheral nerves, non-myelinating Schwann cells ensheath multiple small axons to form Remak bundles (Fig. 6A, B). Remak bundles in tbl nerves contained significantly more axons than control nerves (control: 8.13 ± 1.14; tbl: 12.5 ± 1.73; P = 0.04) (Fig. 6C). Furthermore, the mean axon diameter in tbl was longer than control one (control: 0.66 ± 0.01 μm; tbl: 0.82 ± 0.01 μm; P < 0.001) (Fig. 6D and Supplementary Fig. 1J). Additionally, we observed that in control sciatic nerve (Fig. 6E, F (arrowheads), K) the percentage of axons directly apposed to Schwan cell basal membrane (SCBL) reached the 4.88 ± 1.03%, while it increased 6fold in tbl mice (26.13 ± 7.3%; P < 0.05) (Fig. 6H, J, K). Finally, we observed a 15-fold higher frequency of polyaxonal pockets, considered as evidence for nonmyelinating Schwann cells (NMSCs) anomalous myelination [34, 42], in tbl sciatic nerves as compared with control (control: 0.7 ± 0.39%; tbl: 10.75 ± 1.5%; P < 0.005) (Fig. 6L). In conclusion,
Mol Neurobiol Fig. 4 Electron microphotographs of longitudinal sections through the sciatic nerve of control (A, D, E) and tambaleante (tbl) (B, C, F, G) mice. Instead, the regular successive lamellae disposition of the myelin at the SchmidtLanterman incisures (A, arrows) and the Ranvier node (D, E), tbl axons show hypertrophic myelin infoldings (B, C, arrows; F, G) that compress the axon and reduce the nodal space (compare E, G). In addition, anomalous hypertrophic myelin infoldings (C, F, asterisks) and thicker myelin sheath with degenerative myelin signs (F, arrowheads) are observed. a, axon . S-L, SchmidtLanteman incisure. Scale bars: 5 μm (D), 2 μm (A–C, F), and 1 μm (E, G)
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Akt Regulates Axon Wrapping and Myelin Sheath Thickness Alteration in tbl Nerve Terminals It has been previously reported that HERC1 interacts with TSC2 (tuberous sclerosis complex). TSC1-TSC2 complex is a negative regulator of mTORC1 (mammalian target of rapamycin complex 1). Indeed, the mTORC1 function is upstream controlled by PI3K/Akt [43]. A role of Akt protein in axon wrapping and myelin sheath thickness has been recently proposed [44]. Therefore, we hypothesized that the alterations in the myelination of the tbl axons correlate with a dysregulation in Akt. In order to clarify this issue, we analyzed the amount of phosphorylated Akt in the sciatic nerve. We found an approximately fourfold increase of phosphorylated (Ser473) Akt2 in tbl sciatic nerve as compared with controls [control: 51.3 ± 14.3%, (N = 7); tbl: 205.9 ± 44.7%, (N = 9); P = 0.01] (Fig. 7A; left graph). In contrast, no significant differences in phosphorylated Akt1/3 proteins were found between groups [control: 93.1 ± 15.9%, (N = 7); tbl: 79.8 ± 11.2%, (N = 9); P = 0.5] (Fig. 7A; right graph). Likewise, neither western blot (Fig. 7B) nor RT-PCR (Fig. 7C) techniques revealed variations in dephosphorylated Akt between the two groups. To verify specific disruption of the Akt signal, we estimated Erk protein levels in their dephosphorylated (Fig. 7D) and phosphorylated (Fig. 7E) form. In both cases, no significant differences were found between controls and
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G mutants. Finally, to confirm the mTOR downregulation previously described by Mashimo et al. [1] in brain and kidney, we tested the phosphorylated state of S6K1, substrate of the mTORC1 activity. We observed a decrease of the phosphorylation of S6K1 at threonine 389 (P-T389-S6K1) in sciatic nerves of mutant mice [control: 271 ± 75.4%, (N = 3); tbl: 84.6 ± 23.3%, (N = 5); P = 0.013] (Fig. 7F).
Discussion We present compelling evidence that HERC1 E3 Ubiquitin Ligase plays an important role in the peripheral motor nervous system by maintaining normal conduction of action potentials along the axon. The mutation of HERC1 E3 Ubiquitin Ligase gene (Gly483Glu substitution) in tbl mutant mice provokes a progressive lengthening of the latency of evoked release. We also show that there is a mislocalization of the somatic processes of the tSCs that invade the NMJ synaptic cleft. Besides, we found clear indications of alterations in axon myelination and sheathing accompanied by an increase of Akt phosphorylation in the tbl sciatic nerve. Our previous results showed how the mutation located in the HERC1 gene alters neurotransmitter release and contributes to the modified motor phenotype in the tbl mouse. Importantly, this phenomenon precedes cerebellar deterioration [5]. The current work demonstrates that, as it has been previously observed in other mouse models with neuromuscular pathology [26, 45, 46], the evoked muscular response is
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Fig. 5 Electron microphotographs of transverse sections through the tbl sciatic nerve. Thicker myelin sheaths show aberrant signs of myelin structure consisting in tomacula (B, arrowhead) and aberrant hypermyelination foci (A–M, asterisks) whose growth protrudes within the axon (B–D, J–L, arrows) and compresses the axon cytoplasm. The myelin infoldings within the axons (C–F, J, M) appear as islands of degenerating myelin within (F, J, arrows). Medium size myelinated axons seem to be less affected (A, G); however, alterations of the outer (H, arrow) and inner mesaxon (A, arrow) are frequently detected. The
number of the major dense lines (N, double arrows, 32 in this axon) does not vary, while the intraperiod line is greater in the myelin infolding (N, arrowheads). Hypermyelination foci ratio is higher in tbl section (O). The g ratio is higher in control than in tbl small and medium size axons; however, g ratio value differences remain below the level of statistical significance in the great diameter axons (P). (Q) Scatter plot of g ratio vs axon diameter from control and tbl mice (N = 100 axons/genotype). a, axon. Scale bars: 2 μm (D, J), 1 μm (A–C, G, M), 0.5 μm (E, F; I, K, L), and 0.2 μm (H, N). ***P < 0.0005
delayed in these mice. Since both myelinating and nonmyelinating Schwann cells are in part responsible for maintaining an efficient conductivity of the axon potential [47], firstly, we focused on the NMSCs present in the NMJs. The somatic processes of tSCs cap the nerve terminal without wrapping the synaptic ending of nerve terminal branches [39]. This plays a crucial role in the regulation of the synaptic homeostasis [48, 49]. Our results show that somatic processes of tSCs in tbl mutant mice triggered an excessive growth through the synaptic space affecting the pre- and postsynaptic terminal (Fig. 2). The tbl mouse is not the only case in which tSCs somatic processes invade the neuromuscular synaptic cleft; in fact, similar interposition of tSCs somatic processes have been found in other mouse models with neuromuscular dysfunction [26, 50–52]. Therefore, the herein observed intrusion of the synaptic cleft could explain the axonal connectivity
delay because the presynaptic and postsynaptic membranes must be in close proximity to perform an efficient relay of information [51]. The myelinating Schwann cells are responsible for axo– glial interactions and for electrically isolation of the axon [53, 54]. The ultrastructural study of the tbl sciatic nerve revealed a decrease of the g ratio. This suggests that in tbl mutant mice, the altered myelin sheaths grew along axons in small and medium size (Fig. 5). This could explain the change in their velocity of conduction. Furthermore, the alteration of myelin wrapping in tbl sciatic nerves seems to be reinforced by the abnormalities found in the ensheathment of axons by NMSCs in Remak bundles (Fig. 6). These abnormalities were similar to those described in other mice models of peripheral neuropathies [34, 42, 44]. Additionally, these structural changes of NMSCs forming Remak bundles, which have been
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H proposed as explanation for the sensory pathology found in peripheral neuropathies [34, 55], could explain the hyperalgesia previously reported in tbl mice [56]. Anomalies in myelination have been described in several mice models [40], including genetically modified mice models of structural components of the myelin (i.e., PMP-22 deficient mice, [57]) and mediators of axon-glial interactions (i.e. LRP1, [34]). The herein found myelin anomalies in the tbl mice model phenocopy important aspects of phosphatase and tensin homolog (PTEN) conditional mice [58, 59], as well as transgenic mice expressing a membrane-targeted, activated form of Akt under control of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase promoter [44]. Interestingly, recent studies have shown that the SCs TSC1/2 complex plays a crucial
+
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Axons direct exposition to SCBL (%)
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Fig. 6 Electron microphotographs of transverse sections through control (A) and tbl (B) sciatic nerve. Nonmyelinating Schwann cells (NMSCs) completely ensheath (+) isolated small axons forming the Remak bundles. The number of axons per Remak fiber (C) and the mean axonal diameter (D) are higher in tbl mutant mice. Unmyelinated axons are independently isolated in the Remak bundles by nonmyelinating Schwann cell cytoplasm processes (+). However, some of them are located directly at the Schwann cell basal membrane (SCBL) (E, F, H, J, arrowheads). The percentage of axons directly apposed to the SCBL are higher in tbl than in control sciatic nerves (K). Poly-axonal pockets (G, H– J, asterisks) are also less frequent in control than in tbl sciatic nerves (L). NMSC, nonmyelinating Schwann cell. Scale bars: 1 μm (A, B, E–H), and 0.5 μm (I, J). *P < 0.05; **P < 0.005
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role in regular peripheral nerve development throughout mTOR [60, 61]. All these studies implicate the PI3K/Akt/ mTOR pathway. Moreover, previous in vitro studies demonstrated that PI3K activation is necessary for proper PNS myelination [62, 63]. The above mentioned results led us to subsequently in vestigate the expression of Akt in tbl sciatic nerves using a Western blot approach. In this context we observed an increase of activated Akt-2, the active form prevalent in Schwann cells [44], that could trigger the myelin alter ation observed in tbl mutant mice. However, it remains unclear how the HERC1 mutation, which causes the tbl phenotype, is related to Akt signaling. Previous studies described that HERC1 interacts with TSC2 in vitro [43], preventing the formation of the complex TSC1-TSC2, and
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Fig. 7 The Akt-2 phosphorylated form (Akt2-P) is increased in tbl sciatic nerve. Proteins from the sciatic nerve were separated by electrophoresis, transferred to nitrocellulose membranes and stained using the specific antibodies indicated. Akt-P (N = 7 control and N = 9 tbl) (A), Akt (N = 7 control and N = 9 tbl) (B), Erk-P (N = 4 control and N = 3 tbl) (D), Erk
(N = 4 control and N = 3 tbl) (E), and S6K1-P (N = 3 control and N = 5 tbl) (F). Western blot results are expressed as percentage of intensity relative to GAPDH bands. (C) Expression level of Akt by RT-PCR (N = 9 control and N = 9 tbl). GAPDH expression was used as housekeeping. *P < 0.05
thus inhibiting mTOR function. Mashimo and collaborators reported that tbl mutant mice showed a drop of phosphorylated S6K1 in the brain. This observation may explain the impairment of mTOR signaling in the present mouse model [1], where similar results were found in the sciatic nerve (Fig. 7). In addition, mTOR also controls the level of autophagy and consequently, an increment of autophagy function in tbl mutant mice [1, 31]. Therefore, we hypothesize that a possible compensatory mechanism works throughout mTOR and S6K1 (Fig. 8), generating an increment in phosphorylated Akt in Schwann cells of the tbl sciatic nerve. Alternatively, HERC1 may control the level of phosphorylated Akt directly or with other intermediaries, i.e., mutated HERC1 could stabilized the phosphorylated form of Akt-2 (Fig. 8); however, further studies are warranted to clarify this issue. Finally, our results suggest that tbl mice could be a potential disease model for peripheral neuropathies. The myelin abnormalities found in our model are histological hallmarks of some CMT forms [64–67] and tomacular-neuropathies [57, 68]. Indeed, CMT mice
models present slowly progressive weakness and atrophy of the distal limb muscles [69, 70], similar to that found in tbl mice [5]. In addition, our findings highlight the possible importance of HERC1 in the development of other neurodegenerative diseases. In fact, recent studies have described HERC1 mutations in patients diagnosed of macrocephaly, dysmorphic facies, and psychomotor retardation (MDFPMR) disorder (OMIM: # 617011) [71–74]. One of the main features of these diseases is a severe decline in motor development and presence of moderate-to-severe hypotonia [73], thus mimicking those alterations described in tbl mutant mice [5]. Therefore, alterations in genes whose products are directly or indirectly interacting with HERC1 (Fig. 8) may share a similar peripheral neuropathic phenotype and are candidates for further investigations. In conclusion, the results of the present study show that HERC1 E3 Ubiquitin Ligase is involved in correct axon myelination as well as NMJ synaptic transmission. It could be hypothesized that the mutation could be responsible for the
Mol Neurobiol Fig. 8 Potential pathway of HERC1-dependent Akt regulation. HERC1 interacts in vitro with TSC (tuberous sclerosis complex) 2 [43] (1), and prevents the complex TSC1-TSC2 formation which inhibits mTOR function. Consequently, in tbl mice, a decrement in the amount of S6K1 phosphorylated protein, as well as an increment of autophagy function, is observed, both of them regulated by mTOR [1] (2). [1, 31] (3). Therefore, we hypothesize that there is a compensatory mechanism throughout mTOR and S6K1 that generates an increment Akt phosphorylation in Schwann cells of the sciatic nerve in tbl mutant mice responsible for this phenotype
compensatory increase of Akt-2 in peripheral nerves provoking the myelin anomalies found in the tbl mice. Finally, our findings suggest tbl mice as a potential animal model for the study of diseases involving peripheral neuropathies. Acknowledgements This work was supported by grants to AMC and RR from the Fundación Ramón Areces—Spain—and DGICYT—Spain (Departamento Gubernamental de Investigaciones Científicas y Tecnológicas: BFU2011-27207); RR [Juan de la Cierva contract JCI2011-08888 from the Ministerio de Economía y Competitividad (MINECO) and VPPI-US from the University of Seville]; JLV and RRL (MINECO: SAF2015-64171-R); JAA (Spanish Junta de Andalucía BIO-122 and DGICYT BFU2015-64536-R), and SB was supported by the Fundación Ramón Areces fellowships; LMR is the recipient of a grant from the Servicio Andaluz de Salud de la Junta de Andalucía (C-0009-2015). We are grateful to Juan Luis Ribas, Cristina Vaquero, and Asunción Fernández for their helpful technical supports (CITIUS, University of Seville) and to Drs. Manuel Sarmiento and Karin Neukam for their helpful comments and for editorial assistance.
Compliance with Ethical Standards Conflict of Interest The authors declare that they have no conflict of interests.
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