1. Introduction and Methods
We published recently in Cells a research article entitled: Opposed Actions of PKA Isozymes (RI and RII) and PKC Isoforms (cPKCβI and nPKCε) in Neuromuscular Developmental Synapse Elimination (by Garcia, N.; Balañà, C.; Lanuza, M.A.; Tomàs, M.; Cilleros-Mañé, V.; Just-Borràs, L.; Tomàs, J. Cells 2019, 8, 1304) (PMID: 31652775) in which we pursued previous investigations on the molecular mechanisms involved in the developmental synapse elimination topic. The present short communication highlights details on the statistical analysis of the differences between the PKA and PKC effects on axonal competition and the synapse loss process.
During the development of the nervous system, synapses formed in excess though only the appropriate connections consolidate. At the neuromuscular junction (NMJ), competition between several motor nerve terminals results in the maturation of only one axon and the elimination of the others [
1,
2]. The activity-dependent release of ACh, adenosine, and neurotrophic factors among other molecules allows the direct mutual influence between motor axon terminals and neurons with the involvement of the postsynaptic muscle and teloglial cells [
3,
4,
5]. Thus, the competitive signaling between motor axons is supported, among other receptors, by presynaptic muscarinic ACh autoreceptors (mAChR: M
1, M
2, and M
4 types), adenosine receptors (AR: A
1 and A
2A), and the tropomyosin-related kinase B neurotrophin receptor (TrkB). In previous studies, we investigated the synergistic and antagonistic relations between these receptors that affect synapse elimination [
6,
7]. Receptors A
1, M
1, and TrkB operate mainly through the protein kinase C (PKC) pathway whereas A
2A, M
2, and M
4 are coupled to the protein kinase A (PKA) pathway [
8]. In the forementioned paper [
9], we described that PKA-I and II activity seems to stabilize multiinervation by delaying both axonal elimination and postsynaptic nicotinic ACh receptors (nAChR) pretzel-like cluster differentiation in P5-P9 neonatal mice. Contrarily, PKC activity promotes both developmental axon loss (through cPKCβI and nPKCε isoform action) and postsynaptic nAChR cluster maturation (a possible role for PKCθ). Thus, a metabotropic receptor-driven balance between PKA and PKC activities in the competing axon terminals and, probably, in the postsynaptic site, could be relevant in developmental synapse elimination phenomenom. The phosphorylation of pre- and postsynaptic PKA and PKC targets involved in transmitter release and nerve terminal and/or nAChRs stability could realize the final molecular mechanism of synapse loss.
Therefore, to further know the PKA and PKC action on this mechanism, we ask several questions that can be answered by comparing PKA and PKC effects on the nerve terminal loss and nAChRs cluster maturation: What can be the relative contribution of each kinase in developmental synapse elimination? What is more determinant to final synapse elimination: PKA inhibition or PKC activation? A detailed statistical analysis of the differences between the PKA and PKC effects in the synapse elimination process will help to explore these crucial points and encourage more specific experiments. Here we provide this analysis by using the data of the previous paper in
Cells. Experiments were performed on the Levator auris longus (LAL) muscle from P9 transgenic B6.Cg-Tg (Thy1-YFP)16 Jrs/J mice. In summary, subcutaneous injections of solutions (
Table 1) were administered on the LAL external surface as previously described [
10,
11].
The animals received an injection (50 μL) from P5 to P8 and the LAL muscles were dissected on day P9. Then, after fixation, muscles were incubated with tetramethylrhodamine conjugated α-bungarotoxin (TRITC-α-BTX, 1 h at room temperature; 1:800 dilution of 1 µg/mL; Molecular Probes, Eugene, OR, USA). Analysis of innervation and mAChR maturation were made by using confocal microscopy (
Figure 1). To see the effect of the treatments on the nerve terminals, the number of axons innervating each nAChR receptor cluster were counted. The NMJs were classified in monoinnervated or polyinnervated (innervated by two or more terminal axons). At the same time, the percentage of immature nAChR clusters was defined as the uniform, density-homogeneous nAChR oval plaques, without inhomogeneities in the receptor density or the presence of initial gutters [
12,
13,
14]. Percentages of multiple innervation and immature nAChR receptor clusters were assessed by Fisher’s test and Bonferroni correction. Twelve muscles from six mice were studied for each condition. A minimum of 100 NMJs per muscle were analyzed. The criterion for statistical significance was
p < 0.05. The data are presented as percentages of NMJ ± SD. *
p < 0.05, **
p < 0.01, ***
p < 0.005.
Table 2 and
Table 3 show the comparison of the effects of specific PKA and PKC activators and inhibitors on the percentage of immature multiinnervated synapses and immature postsynaptic nAChR clusters (nAChR with uniform oval plaque).
2. Results and Conclusions
Firstly, we compared PKA activators with PKC inhibitors (both situations delayed synaptic maturation) and we found no difference in either number of axons per synapse or in the morphological postsynaptic maturation (in this last case, only when general PKC inhibitors are used). However, the specific block of cPKCβI or nPKCε with inhibitory peptides (βIV5–3 and εV1–2 respectively) results in no postsynaptic alteration and this is a significatively different situation from PKA stimulation showing the specific presynaptic site of action of these PKC isoforms. In parallel, when comparing PKA inhibitors with PKC activators (the trend of both situations is to accelerate maturation), no difference regarding axonal elimination was found. However, some differences in the nAChR clusters maturation emerged because isoform non-selective PKC activators (mainly PMA) strongly accelerates postsynaptic maturation, suggesting the involvement of another PKC isoform at this site.
Table 2.
Percentage of multiinervation in P9 NMJs.
Table 2.
Percentage of multiinervation in P9 NMJs.
Multiinervation (%) | PKC |
Control | Activator | Inhibitor |
PBS P9 | BRY- 1 (1 nM) | PMA (10 nM) | dPPA (0.2 µg/mL) | FR 236924 (100 nM) | CaC (200 nM) | Che (1 μM) | βIV5–3 (10 μM) | εV1–2 (10 μM) |
41.78 ± 5.61 | 29.16 ± 5.43 | 32.7 ± 2.68 | 26.69 ± 3.02 | 30.59 ± 6.32 | 77.00 ± 6.11 | 68.17 ± 8.21 | 73.64 ± 4.54 | 79.98 ± 9.44 |
PKA | Control | PBS P9 | 41.78 ± 5.61 | - | * | * | * | * | *** | *** | *** | *** |
Activator | Sp8Br (10 μM) | 77.31 ± 5.13 | *** | *** | *** | *** | *** | ns | ns | ns | ns |
Inhibitor | H89 (5 μM) | 32.11 ± 2.53 | *** | ns | ns | ns | ns | *** | *** | *** | *** |
Rp8-Br (100 μM) | 35.03 ± 4.76 | ns | ns | ns | ns | ns | *** | *** | *** | *** |
Rp8-Br (300 μM) | 31.05 ± 3.37 | * | ns | ns | ns | ns | *** | *** | *** | *** |
Rp-cAMPs (100 μM) | 32.04 ± 6.28 | ns | ns | ns | ns | ns | *** | *** | *** | *** |
Rp-cAMPs (300 μM) | 39.24 ± 5.30 | ns | ns | ns | ns | ns | *** | *** | *** | *** |
Table 3.
Percentage of immature AChR postsynaptic clusters in P9 NMJs.
Table 3.
Percentage of immature AChR postsynaptic clusters in P9 NMJs.
Immature clusters (%) | PKC |
Control | Activator | Inhibitor |
PBS P9 | BRY-1 (1 nM) | PMA (10 nM) | dPPA (0.2 µg/mL) | FR 236924(100 nM) | CaC (200 nM) | Che (1 μM) | βIV5–3 (10 μM) | εV1–2 (10 μM) |
9.31 ± 1.73 | 4.76 ± 1.16 | 1.65 ± 0.47 | 8.79 ± 1.37 | 5.92 ± 1.25 | 23.30 ± 4.85 | 16.96 ± 5.61 | 11.10 ± 5.76 | 7.14 ± 3.36 |
PKA | Control | PBS P9 | 9.31 ± 1.73 | - | *** | *** | ns | ns | *** | ** | ns | ns |
Activator | Sp8Br (10 μM) | 28.57± 6.04 | *** | *** | *** | *** | *** | ns | ns | ** | *** |
Inhibitor | H89 (5 μM) | 5.60 ± 2.12 | ns | ns | *** | ns | ns | *** | *** | * | ns |
Rp8-Br (100 μM) | 10.05 ± 2.59 | ns | ns | *** | ns | ns | *** | ns | ns | ns |
Rp8-Br (300 μM) | 16.74 ± 1.09 | * | *** | ** | * | ** | ns | ns | ns | ** |
Rp-cAMPs (100 μM) | 4.70 ± 1.97 | * | ns | *** | ns | ns | *** | ** | ** | ns |
Rp cAMPs (300 μM) | 8.57 ± 3.26 | ns | ns | *** | ns | ns | *** | * | ns | ns |
Second, when comparing the effects of PKA and PKC activators, a significant difference is always observed when looking both for the number of axons per synapse and for the postsynaptic maturation (the significance is always p < 0.005, Fisher’s test) indicating the complementarity of the kinases. Finally, when comparing the effects of PKA and PKC inhibitors a significant difference is always observed when looking for the number of axons per synapse (the significance is also always p < 0.005). However, the comparisons in relation with the postsynaptic maturation show some complexity. Specifically, as stated above, PKA inhibition (with H-89 or some specific RI or RII blockers) barely induces a small change on the postsynaptic clusters, indicating that pharmacologic PKA inhibition above the physiological situation cannot be increased. On the other hand, and similarly, the specific blockers of cPKCβI or nPKCε do not affect nAChRs clusters because of their presynaptic site of action. Thus, it seems that, in the synapse elimination process at the considered developmental period (P9), PKCs may have a significative role in determining maturation as their experimental inhibition strongly affects the process. However, endogenous PKA activity seems to be inhibited during the synapse elimination process because the additional pharmacological inhibition only results in a presynaptic change. Thus, PKA would be not much further additionally inhibited and PKC would be not much further activated, suggesting there is a well balanced effect between them (a similar level of PKA inhibition and PKC stimulation resulting in synapse maturation). Altogether, these results suggest a well stablished balance between PKC and PKA activity that allows the optimal NMJ maturation progression, being PKC more active than PKA during it.
In summary, there are no significant differences exist between the effects of PKA activators and PKC inhibitors or PKA inhibitors and PKC activators on the rate of developmental axon loss rate, indicating the complementarity of the kinases. Indeed, a similar level of PKA inhibition and PKC potentiation (mainly of the cPKCβI and nPKCε isoforms) would be required during development. On the other hand, the kinase effect in the postsynaptic clusters maturation follows the same pattern of PKA/PKC interaction as in the axons despite that without the involvement of cPKCβI and nPKCε isoforms. These detailed statistical results strongly reinforce the previously published data, indicate a direct reciprocal involvement of the PKA and PKC isoforms, and suggest a complementary or cooperative work of them.
To go further into the issue, the following question can be asked: What can be the precise differential involvement of PKA inhibition and PKC activation in nerve terminals in different stages of competition and elimination or strengthening? We should analyze the hypothesis that this particular configuration of kinase’s activity in supernumerary axons is done to be eliminated.
Author Contributions
N.G., V.C.-M., L.J.-B., M.T., M.D. and A.P.: data collection, quantitative analysis; literature search, data interpretation, abstrat graphic design; V.C.-M.: abstrat graphic; N.G., M.A.L.: statistics; J.T., N.G. and M.A.L.: conception and design, literature search, data interpretation, manuscript preparation. J.T., N.G., and M.A.L. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by Ministerio de Ciencia, Innovación y Universidades, the Agencia Estatal de Investigación (AEI) and the European Regional Development Fund (ERDF) PID2019-106332GB-I00, the support of the Universitat Rovira i Virgili (URV) (2017PFR-URV-B2-85) and the Catalan Government (2017SGR704). L.J.-B. has been supported by the Universitat Rovira i Virgili (URV) under the framework of the “Programa Martí i Franquès d’ajuts a la investigació. Contractes de personal investigador predoctoral en formació (PMF-PIPF). A.P. has been supported by the Spanish Ministerio de Ciencia Innovación y Universidades (Grant no. PRE2020-092084, project no. PID2019-106332GB-I00). V.C. and M.D. were supported by a grant from MINECO under the framework of the Sistema Nacional de Garantía Juvenil, the European Social Fund (ESF) and the Iniciativa de Empleo Juvenil (IEJ) reference codes: LE1511314-2014PEJ-04 and LE1911587-2019PEJ-04.
Institutional Review Board Statement
The mice were cared for in accordance with the guidelines of the European Community’s Council Directive of 24 November 1986 (86/609/EEC) for the humane treatment of laboratory animals. All experiments on animals have been reviewed and approved by the Animal Research Committee of the Universitat Rovira i Virgili and Generalitat de Catalunya (Reference number: 10760).
Informed Consent Statement
Not applicable.
Data Availability Statement
We think that our data are not appropriate for the available repository database in neuroscience.
Conflicts of Interest
The authors declare no conflict of interest.
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