The HERC1 ubiquitin ligase regulates presynaptic membrane dynamics of central synapses

1 Vol.:(0123456789) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports The HERC1 ubiquitin ligase regulates presynaptic membrane dynamics of central synapses Mª Angeles Montes‑Fernández1,4, Eva Mª Pérez‑Villegas2,4, Francesc R. Garcia‑Gonzalo 3, Leonardo Pedrazza 3, Jose Luis Rosa3, Guillermo Alvarez de Toledo1* & José A. Armengol 2* HERC1 is a ubiquitin ligase protein, which, when mutated, induces several malformations and intellectual disability in humans. The animal model of HERC1 mutation is the mouse tambaleante characterized by: (1) overproduction of the protein; (2) cerebellar Purkinje cells death by autophagy; (3) dysregulation of autophagy in spinal cord motor neurons, and CA3 and neocortical pyramidal neurons; (4) impairment of associative learning, linked to altered spinogenesis and absence of LTP in the lateral amygdala; and, (5) motor impairment due to delayed action potential transmission, decrease synaptic transmission efficiency and altered myelination in the peripheral nervous system. To investigate the putative role of HERC1 in the presynaptic dynamics we have performed a series of experiments in cultured tambaleante hippocampal neurons by using transmission electron microscopy, FM1‑43 destaining and immunocytochemistry. Our results show: (1) a decrease in the number of synaptic vesicles; (2) reduced active zones; (3) less clathrin immunoreactivity and less presynaptic endings over the hippocampal main dendritic trees; which contrast with (4) a greater number of endosomes and autophagosomes in the presynaptic endings of the tambaleante neurons relative to control ones. Altogether these results show an important role of HERC1 in the regulation of presynaptic membrane dynamics. HERC1 is a giant phylogenetically conserved ubiquitin ligase of the HECT family1 that participates in the ubiquitin–proteasome system (UPS)2–3. Like other UPS alterations, mutations in HECT E3 ligases have been associated with the pathogenesis of neuromuscular disorders, Parkinson’s disease and diseases of the autism spectrum1–4. Furthermore, mutations in the RCC1 domain of human HERC1 have been related with X-linked retinitis pigmentosa and juvenile amyotrophic lateral sclerosis 25. In humans, missense mutations of Herc1 display polymorphic syndromes with or without cerebellar affectation6–8, in which the intellectual disability appears as the common neurological disorder8. The tambaleante (tbl) mutant mouse was earliest reported as a model of adult cerebellar ataxia caused by the almost complete autophagy cell death of cerebellar Purkinje cells9–11. In addition to adult cerebellar Purkinje cell degeneration5,9–12, other alterations in the central and the peripheral nervous system have been recently described in tbl mouse such as: (1) increase of autophagy signs in spinal cord motor neurons and neocortical and CA3 hippocampal pyramidal neurons13; (2) impairment of the associative learning associated to absence of long term potentiation (LTP), altered dendritic spinogenesis, and a drastic decrease of glutamatergic innervation of the lateral amygdala14; (3) anomalous myelination in the sciatic nerve together with alterations of non-myelinating terminal Schwann cells at the neuromuscular junction (NMJ)15; and, (4) altered motor performance owing to a reduction of the motor end-plate area, and impaired evoked neurotransmitter release at the NMJ16. Molecular studies identified that HERC1 mutation carried by the tbl mice, and responsible for autophagy cell death, is a Gly483Glu spontaneous substitution located in the N-terminal RCC1 domain (RLD1)5. This domain acts as guanine nucleotide-release factor for ARF proteins and influences intracellular vesicle trafficking interacting with ARF/Rab GTPases1–2. Furthermore, C-terminal RCC1 domain (RLD2) of HERC1 forms a ternary complex with clathrin (CLT) and the heat shock protein 70, and might influence intracellular vesicular trafficking17. open 1Department of Medical Physiology and Biophysics, School of Medicine, University of Seville, Seville, Spain. 2Department of Physiology, Anatomy and Cell Biology, University Pablo de Olavide, Seville, Spain. 3Department of Physiological Sciences, IDIBELL, University of Barcelona, Barcelona, Spain. 4These authors contributed equally: Mª Angeles Montes-Fernández and Eva Mª Pérez-Villegas. *email: [email protected]; jaarmbut@ upo.es Content courtesy of Springer Nature, terms of use apply. Rights reserved 2 Vol:.(1234567890) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ These data together strongly suggest that HERC1 mutation of tbl mouse might alter the normal dynamic of excitatory presynaptic terminals. Furthermore, CLT mediated endocytosis (CME) is a key step for synaptic vesicle recycling18; thus, alterations of HERC1-CLT interaction1,17 might alter the normal CLT cycle interfering with the normal synaptic function. Therefore, to elucidate the putative role of HERC1 in the synaptic vesicle populations of central excitatory synapses and in their presynaptic dynamics, we have analyzed tbl hippocampal neuronal cultures invitro by using transmission electron microscopy, immunocytochemical, GFP pull-down and FM1-43 destaining methods. Results In present experiments those synapses of control (Fig.1A) and tbl (Fig.1B) hippocampal cultures showing a clear synaptic cleft, and evident thickening of preand postsynaptic zones were only considered for vesicle counts and active zone length measurement. The number of round and clear synaptic vesicles counted and the active zone length were significantly fewer (Fig.1C) and shorter (Fig.1D) in tbl synapses relative to control ones. However, significant differences were neither found in the mean diameter of the synaptic vesicles (Fig.1E,∅) nor in the intervesicular distance (Fig.1E) between control and tbl synapses. Furthermore, there was no statistically significant differences in the number of tethered synaptic vesicles (Fig.1G); and, although the numbers of vesicles located in the nearest (< 75nm) and farthest (225–300nm) compartments of tbl synapses were fewer than in control ones, their values were not significant (p = 0.0853 and p = 0.2318 respectively) (Fig.1H). However, a clear significant statistical difference in the number of docked vesicles was found, whose number was ever higher in control synapses relative to in tbl ones –as much in absolute values (Fig.1F, AZ) as in normalized values relative the mean value of tbl active zone values (Fig.1F, 500nm AZ). To quantify synaptic vesicles pools we use the fluorescence lipophilic dye FM1-43, that label synaptic boutons in an activity-dependent manner that involves dye uptake by synaptic vesicles that are recycling19. Hippocampal cultures of control and tbl mutants were loaded with FM1-43 dye after the application of 600 pulses (Fig.2A and D). The amount of FM1-43 loaded depends on the recycling activity of the neuron. Control cultured neurons revealed a greater amount of fluorescence as compared to tbl ones (450 ± 40; n = 6, vs 250 ± 35; n = 5) indicating larger dye loading. Unloading of FM1-43 of individual boutons for control and tbl revealed a large variability in both animals, reflecting a diversity of individual boutons in the amount of dye loaded (Fig.2B and E). A set of 40 and 700 pulses were sufficient to unload dye completely in both control and tbl cultured neurons (Fig.2C and F). A comparison of the destaining of FM1-43 for the control and tbl is shown in Fig.2G. Quantification of the fluorescence change upon different levels of stimulation is shown in Fig.2H. The application of 40 pulses produced a decrease of 45.7 ± 20.4 fluorescence units in the control (n = 4) versus 16.1 ± 8.9 in the tbl mutant (n = 4). This fluorescence change corresponds to approximately 9 vesicles in the RRP for the control and 3 for the tbl mutant, since previous experiments in the same set up revealed 5 fluorescence units per synaptic vesicle20. The application of 700 consecutive pulses, sufficient to deplete the reserve pool, showed also a significant difference between the control and the tbl mutant hippocampal cultured neurons (238.9 ± 108 vs. 122 ± 51.3; n = 100 and n = 75 boutons, p = 0.007). Cultured tbl hip21pocampal neurons seem not show evident qualitative differences in size relative to control ones. The use of microtubule associated protein 2 (MAP2) as neuronal dendritic marker allowed us to analyze the neuronal expression of CLT (Fig.3A-B). Control hippocampal neurons expressed strongly both markers relative to tbl ones; and the quantitative analyses demonstrated the existence of significant differences in the absolute (Fig.3C and E) and normalized values relative to the analyzed area (Fig.3D and F) of MAP2 and CLT expression. As CLT plays a key role in the synaptic vesicle endocytic recycling22, we have analyzed the immunocytochemical expression of CLT together to the presynaptic SV2B protein as general marker of synaptic vesicles (Fig.4A-B). The expression of both markers was significantly higher in control cultured neurons than in tbl ones (Fig.4CF). Moreover, the counts of presynaptic boutons expressing SV2B alone or co-expressing with CLT (Fig.5A-B) clearly indicated that the number of presynaptic boutons lying over the main dendrites of control hippocampal neurons analyzed here was higher than those ending on the main dendrites of tbl hippocampal neurons (Fig.5C, T); this significant difference in the number of synaptic boutons is consistent from both SV2B single (Fig.5C, SV2B) and double labelled boutons (Fig.5C). When these numbers were normalized regarding their density relative to each µm of dendritic shaft measured, difference clearly ratifies that the density of synaptic boutons ending on the main dendrite of tbl hippocampal neurons was the half in relation to the control (Fig.5E). The reduction in the density of presynaptic boutons is also correlated with a significant decrease in the immunolabelling of synaptotagmin 1 (SYT1), a presynaptic ubiquitous protein22, in tbl cultured neurons in relation to control cultured neurons (Fig.6D). In our hands the commercial antibody used here against the HERC1 in hippocampal cultures (Figs.6 and 7) shows that the expression of this protein is distributed through the cell nucleus and the cytoplasm (Figs.6A-B and 7A-B). In all analyzed cultures, HERC1 expression was significantly more intense in tbl hippocampal neurons than in controls (Figs.6C and 7C). Apart the differences in the number of rounded vesicles, an also clear qualitative difference on the size of the endosomes was observed between tbl and control presynaptic endings (Fig.8A to D). From seminal works of Heuser and Reese23 the endosomes located in the presynaptic endings have been related with the synaptic vesicles recycling. In present observations, quantitative analysis of the main components of the CLT recycling pathway24–25 demonstrated statistic significant differences on the number of CLT coated vesicles, endocytic pits, and endosomes. Thus, while control presynaptic terminals contained more coated vesicles, and endocytic pits than tbl ones (Fig.8E); the number of endosomes was greater in tbl presynaptic boutons relative to control ones (Fig.8E). Moreover, the qualitative appearance that tbl endosomes were bigger than control ones is clearly ratified by the measurement of their maximum diameters that were twice in tbl presynaptic endings relative to the Content courtesy of Springer Nature, terms of use apply. Rights reserved 3 Vol.:(0123456789) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ Figure1. Electron microscopy microphotographs illustrating the ImageJ counts of the number of synaptic vesicles in control (A) and tbl (B) hippocampal cultures. The active zone is consistently shorter in tbl than in control presynaptic boutons (D; *p = 0.0246). Quantitative analyses demonstrate that tbl synapses possess less synaptic vesicles than control ones (C; *p = 0.0391), and fewer docked synaptic vesicles in both absolute (F AZ, *p = 0.0470) and normalized values (F; 500nm AZ, *p = 0.0265). However, no significant statistic differences are found in the synaptic vesicles diameter (E; ∅, p = 0.1452), the intervesicular distance (E, iV distance, p = 0.2184), in the number of tethered vesicles (G; AZ, p = 0.2011; 500nm AZ, p = 0.0834), and in the number of vesicles respecting their distance from the active zone (H; < 75nm, p = 0.0853; 75–150nm, p = 0.4834; 150–225nm, p = 0.4342; 225–300nm, p = 0.2318). Bars = 200nm. Content courtesy of Springer Nature, terms of use apply. Rights reserved 4 Vol:.(1234567890) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ control ones (Fig.8F). This dysregulation of the recycling endocytosis pathway is reinforced by present immunocytochemical experiments. Early endosome antigen 1 (EEA1) distributed through the neuronal cytoplasm (Fig.7A-B); and the quantitative analysis shows significant differences of the immunocytochemical expression of EEA1 between tbl and control cultured hippocampal neurons, being almost twice in tbl neurons regarding the control ones (Fig.7D). Another important finding of present experiments is related with the presence of autophagy vacuoles. Thus, from earliest reports, the ultrastructural features of these vacuoles as it is the presence of double membrane encircling damaged or ubiquitinated proteins26 help their easy identification. Autophagosomes and mitochondrial debris (Fig.9B) are often observed in tbl presynaptic boutons; and, in addition to the previously described Figure2. FM1-43 destaining of control and tbl hippocampal neurons in culture with successive applications of 40 APs, to deplete the RRP, and 100 plus 600 APs to deplete the reserve pool of synaptic vesicles. (A), control neuron stained with FM1-43. The ROIs are shown in red. (B), time course of FM1-43 destaining for multiple boutons. (C), Average FM1-43 destaining (left axis normalized fluorescence, right axis total fluorescence. (D), tbl hippocampal neuron stained with FM1-43. ROIs marked in red as in (A). (E,F), time course of FM143 destaining for tbl hippocampal neurons for individual boutons (E) and the average response (F). (G), superposition of control and tbl FM1-43 response for comparison. Note that control neurons were better loaded than tbl mutant neurons. (H), both, the RRP and the size of the reserve pool of synaptic vesicles were smaller in the tbl mutant, suggesting a lower size of dock and recycle vesicles. The asterisks indicate significant differences in the Student’s t test: ***p = 0.04). Content courtesy of Springer Nature, terms of use apply. Rights reserved 5 Vol.:(0123456789) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ differences in the endosomes’ number and diameters between control cultured hippocampal neurons and tbl ones; the values of the counts of these vesicles showed an important increment of their number within tbl Figure3. Laser confocal microphotographs of hippocampal cultures from control (A), and tbl (B) mice illustrative of MPA2 (green) and CLT (red) immunocytochemistry. (C–F), graphical representation of the fluorescence intensities of MAP2 and CLT in absolute values per image (C,E); and relative to the % of measured area (D,F). In both absolute (C,E) and relative (D,F), values, MAP2 and CLT expressions are higher in control cultures than in tbl ones. The asterisks indicate significant differences in the Student’s t test: ***p = 0.0008 (C); **p = 0.005 (D); ***p = 0.0005 (E); and ***p = 4.4E−06 (F). Bars = 50µm. a.u., arbitrary units. Content courtesy of Springer Nature, terms of use apply. Rights reserved 6 Vol:.(1234567890) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ presynaptic terminals (n = 26) relative to control ones (n = 30) (Fig.9C). From these observations it seems that the membrane dynamics of the presynaptic terminal might be altered in the tbl mutation. As the synaptic Figure4. Laser confocal microphotographs illustrative of SV2B (green) and CLT (red) immunoreactivities in control (A) and tbl (B) hippocampal cultures. (C–F), graphs showing the fluorescence intensities of CLT (C, D) and of synaptic vesicles identified by SV2A immunoreactivity (E,F). In both absolute and % relative values, fluorescence intensity was higher in control than in tbl cultures (C, **p = 0.004; D, **p = 0.002; E, **p = 0.004; F, **p = 0.003). Bars = 30µm. a.u., arbitrary units. Content courtesy of Springer Nature, terms of use apply. Rights reserved 7 Vol.:(0123456789) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ Figure5. ImageJ composites illustrative of the count of presynaptic boutons expressing SV2B (green), CLT (red), or both immunoreactivities in control (A) and tbl (B) hippocampal cultures. (C–E), graphs showing the number of single SV2B and double (SV2B-CLT) labelled boutons (C), the percentage of labelled boutons (D), and their density of counted boutons per dendrite µm (E). The number of presynaptic boutons is higher in control relative to tbl cultures (C, T: *p = 0.014; SV2B-CLT: **p = 0.005; SV2: *p = 0.04). Correlative to these counts, the percentage of double labelled boutons is lower in tbl cultures relative to control ones (D, SV2B-CLT: **p = 0.007). The density of the number of all categories of presynaptic boutons counted is ever lower in tbl cultures than in control ones (E, T: **p = 0.005; SV2B-CLT: **p = 0.003; SV2: **p = 0.009), while no significant differences were found on the mean of the dendrite’s length measured (F; p = 0.544). Bars = 5µm. T, total number of counted boutons. Content courtesy of Springer Nature, terms of use apply. Rights reserved 8 Vol:.(1234567890) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ transmission takes place at the terminal active zone, which is smaller in tbl presynaptic terminal than in control ones (Fig.1C-D), we have measured the perimeter of the presynaptic boutons, the perimeter of the endosomes Figure6. Laser confocal microphotographs illustrative of HERC1 (red) and SYT (green) immunoreactivities in control (A) and tbl (B) hippocampal cultures. HERC1 is expressed in both the neuronal nucleus (A–B, n) and cytoplasm (A–B, arrows). (C–D) graphs showing the fluorescence intensities of HERC1 (C) and of presynaptic terminal endings identified by SYT (D). Herc1 expression is greater in tbl cultured neurons relative to control ones (C, *p = 0.04723). In contrast, SYT immunoreactivity is considerably higher in control cultured neurons compared to tbl ones (D, *p = 0.04651). Bars = 5µm. Content courtesy of Springer Nature, terms of use apply. Rights reserved 9 Vol.:(0123456789) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ and the perimeter of the autophagosomes and compared these in absolute values and as their ratios in relation to the active zone length (Fig.9). Thus, while the values of presynaptic boutons perimeters and of the sum of the perimeter of the plasma membrane surrounding the bouton and that of the endoand autophagosomes did not shown significant differences between control and tbl terminals (Fig.9D), the perimeter of endosomes and Figure7. ImageJ composites illustrative of the expression of HERC1 (red) and EEA1 (green), in control (A) and tbl (B) hippocampal cultures. (C–E), graphs show the intensity of the expression of HERC1 (C) and EEA1 (D). HERC1immunoreactivity is higher in tbl relative to control cultures (C, *p = 0.0404). Likewise, EEA1 expression is lower in control cultures relative to tbl ones (D, *p = 0.04276). Bars = 5µm. n, neuronal cell nucleus. Content courtesy of Springer Nature, terms of use apply. Rights reserved 16 Vol:.(1234567890) Scientific RepoRtS | (2020) 10:12057 | https://doi.org/10.1038/s41598-020-68970-8 www.nature.com/scientificreports/ Plasmids and pull‑down experiments. pFG41 construct (GFP-RLD1 fusion containing 365–794 amino acid residues of HERC1) was previously described60. pFG44 construct was similar to pFG41 but containing the punctual mutation Gly483Glu found in tbl animals. This construct was generated from pFG41 using QuickChange XL Site-directed mutagenesis kit (Stratagene) following instructions of manufacturer.For the GFP pulldown, supernatants were incubated with 2μL of GFP-Trap© Agarose resin (Chromotek, Germany) around 2h at 4°C. After the incubation, the samples were centrifuged (2,500g) and pull-downs were washed with NP40 buffer (three times). After each wash the samples were centrifuged again. Pellets were analyzed by electrophoresis and immunoblotting by using the following antibodies: anti-HERC1 (410)55, anti-GFP (Abcam), and anti-clathrin (CLT) heavy chain (hc) (BDBiosciences). Statistical analysis. The statistical analyses of the data from the TEM, FM1-43 destaining, and immunolabelling experiments were analyed blind by EMP-V and MAM-F. A two tailed Student’s t test was used to compare the data from tbl and control hippocampal cultures. Any p-values less than 0.05 were considered significant, indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001. Received: 17 October 2019; Accepted: 3 July 2020 References 1. García-Cano, J., Martínez-Martínez, A., Sala-Gaston, J., Pedrazza, L. & Rosa, J. L. HERCing: Structural and functional relevance of the large HERC ubiquitin ligases. Front. Physiol. 10, 1014. https ://doi.org/10.3389/fphys .2019.01014 (2019). 2. 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Ribas for his assistance at the CITIUS, and Dr I. de Benito for maintaining the animal facilities and the tambaleante mouse line. This work was supported by the following grants: GAT (Spanish Junta de Andalucía BIO-209 and MINECO-DGICYT BFU2015-64536-R); JAA (Spanish Junta de Andalucía BIO-122 and MINECO-DGICYT BFU2015-64536-R); and JLR (MINECO-AEI/FEDER, UE, BFU2016-80295-R). Author contributions Design: J.A.A. and G.A.de.T. Data acquisition, analysis and interpretation: M.A.M.-F., E.M.P.-V., F.G.-G., L.P., J.A.A., and G.A.de.T. Writing-original draft: J.A.A. and G.A.de.T. & J.L.R. Writing-review & editing: M.A.M.-F., E.M.P.-V., J.L.R., G.A.de.T., and J.A.A. Competing interests The authors declare no competing interests. Additional information Supplementary information is available for this paper at https ://doi.org/10.1038/s4159 8-020-68970 -8. Correspondence and requests for materials should be addressed to G.A.T.orJ.A.A. Content courtesy of Springer Nature, terms of use apply. 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