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. 2015 Nov;8(11):1389-400.
doi: 10.1242/dmm.021246. Epub 2015 Sep 3.

Loss of the Coffin-Lowry Syndrome-Associated Gene RSK2 Alters ERK Activity, Synaptic Function and Axonal Transport in Drosophila Motoneurons

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Free PMC article

Loss of the Coffin-Lowry Syndrome-Associated Gene RSK2 Alters ERK Activity, Synaptic Function and Axonal Transport in Drosophila Motoneurons

Katherina Beck et al. Dis Model Mech. .
Free PMC article

Abstract

Plastic changes in synaptic properties are considered as fundamental for adaptive behaviors. Extracellular-signal-regulated kinase (ERK)-mediated signaling has been implicated in regulation of synaptic plasticity. Ribosomal S6 kinase 2 (RSK2) acts as a regulator and downstream effector of ERK. In the brain, RSK2 is predominantly expressed in regions required for learning and memory. Loss-of-function mutations in human RSK2 cause Coffin-Lowry syndrome, which is characterized by severe mental retardation and low IQ scores in affected males. Knockout of RSK2 in mice or the RSK ortholog in Drosophila results in a variety of learning and memory defects. However, overall brain structure in these animals is not affected, leaving open the question of the pathophysiological consequences. Using the fly neuromuscular system as a model for excitatory glutamatergic synapses, we show that removal of RSK function causes distinct defects in motoneurons and at the neuromuscular junction. Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function. Furthermore, loss of RSK function interferes with ERK signaling at different levels. Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse. In addition, we uncovered a novel function of RSK in anterograde axonal transport. Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions. In this context, RSK acts as a modulator of ERK signaling.

Keywords: Axonal transport; Drosophila; MAPK signaling; Motoneuron; Neuromuscular junction; RSK; Synapse.

Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Localization of RSK at the presynapse. Left panels: projection view of a neuromuscular junction at muscle 6/7 stained for Bruchpilot (BRP; red), glutamate receptor II subunit D (GluRIID; blue) and transgenic GFP::RSK (green) expressed in motoneurons with the driver line D42-Gal4. Scale bar: 10 µm. Right panels: detail of a single bouton shown in the red box. A single confocal section is shown. BRP marks several active zones in the bouton, which are apposed by GluR fields marked by GluRIID. Presynaptic GFP::RSK staining is also seen outside active zones. Scale bar: 2 µm.
Fig. 2.
Fig. 2.
Localization of RSK and pERK in motoneurons. (A) Left panels: projection view of a neuromuscular junction at muscle 6/7 stained for GFP::RSK expressed with D42-Gal4 (green), phosphorylated ERK (pERK; red) and horseradish peroxidase (HRP; blue). Scale bar: 20 µm. Right panels: single confocal section of a bouton shown in the red box. GFP::RSK and pERK partly colocalize at presynaptic sites. Scale bar: 2 µm. (B) Staining of the soma of a motoneuron (outlined by dashed line) for GFP::RSK (green), pERK (red) and Lamin (blue) as a nuclear membrane marker. RSK and pERK localize to the perikaryon. In addition, pERK staining is also seen in the nucleus. Scale bar: 2 µm.
Fig. 3.
Fig. 3.
Loss of RSK increases ERK activity. (A) Western blots of lysates from third larval instar ventral ganglia probed with antibodies against total ERK, phosphorylated ERK (pERK) and α-tubulin (α-tub). The ERK antibody detects non-phopshorylated (arrowhead) and phosphorylated ERK (open arrowhead). (B,C) Quantification of ERK (B) and pERK (C) levels normalized to α-tubulin from at least five independent biological experiments (denoted in the bars). Compared with wild type, the level of pERK but not of ERK is increased in RSKΔ58/1 and returned to wild-type levels in the presence of the P[RSK] transgene. *P≤0.05 and ***P≤0.001.
Fig. 4.
Fig. 4.
Loss of RSK affects pERK levels and subcellular distribution in motoneurons. (A) Somata of motoneurons in controls or RSKΔ58/1 animals were identified by D42-Gal4-driven expression of the mCD8::GFP marker. Motoneuron somata are outlined by dashed lines. Staining for GFP (green), Lamin (blue) and phosphorylated ERK (pERK; red) showed increased pERK signals in the perikaryon and nucleus of RSKΔ58/1 motoneurons. Scale bar: 2 µm. (B,C) Left panels: projection view of neuromuscular junctions (NMJs) from wild type (B) and RSKΔ58/1 (C) stained for Bruchpilot (BRP; green), pERK (red) and horseradish peroxidase (HRP; blue), which labels the complete neuronal terminal. Scale bar: 20 µm. Right panels: single confocal sections of boutons (red boxes) are shown in detail. Compared with wild type (B), staining of pERK is nearly depleted at the NMJ of RSKΔ58/1 animals (C). Scale bar: 2 µm. (D,E) pERK signal intensities were quantified at the NMJ (D) and in cell bodies (E) from whole central nervous system and body-wall preparations from control and RSKΔ58/1 animals and normalized to mCD8::GFP expressed in motoneurons (see Fig. S2). The number of preparations analyzed is denoted in the bars. **P≤0.01.
Fig. 5.
Fig. 5.
Effects of RSKΔ58/1 on neuromuscular junction size, active zones and receptor fields. Quantifications were done at the neuromuscular junctions (NMJs) of muscle 6 and 7 in abdominal segment A2 or A3. (A) NMJ size was determined by outlining horseradish peroxidase (HRP) staining. (B,C) Bruchpilot (BRP) was used to quantify the number (B) and area (C) of active zones per NMJ. (D,E) Glutamate receptor II subunit D (GluRIID) was used to determine the number (D) and area (E) of glutamate receptor (GluR) fields per NMJ. Compared with wild type, all measured parameters are significantly reduced in RSKΔ58/1. (F) The ratio of BRP to GluRIID is depicted and does not differ between genotypes. NMJ size and presynaptic defects of RSKΔ58/1 are rescued by P[RSK], whereas postsynaptic defects are not rescued or partly rescued. (A-F) Animals homozygous for the hypomorphic ERK allele rl1 exhibit wild-type NMJ morphology. By contrast, the activated variant rlSem caused a significant decrease in NMJ size (A), number of active zones (B) and GluR fields (D). The number of individual larvae per genotype analyzed is denoted in the bars. *P≤0.05, **P≤0.01 and ***P≤0.001.
Fig. 6.
Fig. 6.
Electrophysiological characterization of RSK mutant synapses. (A) Example traces and quantification of miniature excitatory junctional currents (minis) recorded in two-electrode voltage-clamp recordings from the larval neuromuscular junction. The average mini amplitude was significantly smaller in RSKΔ58/1 and could not be restored by employing a genomic rescue construct (RSKΔ58/1;P[RSK]). Mini frequency was not affected by loss of RSK. (B) Representative evoked excitatory postsynaptic currents (eEPSCs; stimulation artifact removed for clarity) during low-frequency nerve stimulation (0.2 Hz) and quantification of amplitudes. (C) Quantal content was comparable in all genotypes. Scale bars: (A) 2 nA (nanoampere), 50 ms; (B) 20 nA, 10 ms. **P≤0.01 and ***P≤0.001.
Fig. 7.
Fig. 7.
Loss of RSK causes proximal accumulations of synaptic proteins and transport defects. (A) Motoneuron axons from wild type, RSKΔ58/1 and RSKΔ58/1;P[RSK] were stained for Bruchpilot (BRP; upper row) and cysteine string protein (CSP; lower row). Scale bar: 20 µm. (B,C) Quantifications for BRP (B; n=5 individual larvae per genotype) and CSP particles (C; n=8 individual larvae per genotype). (D) Analysis of the axonal transport of mitochondria in motoneurons by in vivo imaging. After bleaching of a length of axon, the movement of mitochondria was recorded in anesthetized larvae. Plots display anterograde- and retrograde-transported and stationary mitochondria in control animals (OK6-Gal4;UAS-mito::GFP, n=15) and the RSK mutant (RSKΔ58/1;OK6-Gal4;UAS-mito::GFP, n=10). *P≤0.05 and **P≤0.01.

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