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Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons.

Beck K, Ehmann N, Andlauer TF, Ljaschenko D, Strecker K, Fischer M, Kittel RJ, Raabe T - Dis Model Mech (2015)

Bottom Line: Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function.Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse.Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions.

View Article: PubMed Central - PubMed

Affiliation: University of Würzburg, Institute of Medical Radiation and Cell Research, Versbacherstraße 5, Würzburg D-97078, Germany.

No MeSH data available.


Related in: MedlinePlus

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.
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DMM021246F3: 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.

Mentions: Using a previously characterized mutant for RSK (RSKΔ58/1; Putz et al., 2004), we next determined whether complete loss of RSK alters overall levels of pERK. To enrich for the population of motoneurons, we restricted the analysis to the ventral ganglion. Ventral ganglia were dissected from third instar larval central nervous system preparations, and lysates were analyzed by western blot using antibodies against ERK and pERK, with α-tubulin for normalization (Fig. 3A). Normalized ERK levels showed no difference between wild-type and RSKΔ58/1 preparations (Fig. 3B; wild type=1.00±0.12; RSKΔ58/1=1.02±0.11), indicating that loss of RSK has no influence on overall ERK levels. By contrast, pERK levels were significantly increased in RSKΔ58/1 compared with wild type (Fig. 3C; wild type=1.00±0.14; RSKΔ58/1=1.25±0.16, P<0.001). To validate the observed changes in pERK activity, a 20 kb genomic rescue construct encompassing the 6 kb RSK transcription unit (in the following, termed P[RSK]) was introduced into a RSKΔ58/1 background. In RSKΔ58/1;P[RSK] animals, normalized pERK levels became significantly different from RSKΔ58/1 (Fig. 3A,C; 1.02±0.15, P<0.05 versus RSKΔ58/1) and returned to wild-type levels. In summary, the results are in line with previous genetic analyses, which indicated a function of RSK as a negative regulator of the ERK/MAPK signaling pathway (Kim et al., 2006).Fig. 3.


Loss of the Coffin-Lowry syndrome-associated gene RSK2 alters ERK activity, synaptic function and axonal transport in Drosophila motoneurons.

Beck K, Ehmann N, Andlauer TF, Ljaschenko D, Strecker K, Fischer M, Kittel RJ, Raabe T - Dis Model Mech (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4631788&req=5

DMM021246F3: 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.
Mentions: Using a previously characterized mutant for RSK (RSKΔ58/1; Putz et al., 2004), we next determined whether complete loss of RSK alters overall levels of pERK. To enrich for the population of motoneurons, we restricted the analysis to the ventral ganglion. Ventral ganglia were dissected from third instar larval central nervous system preparations, and lysates were analyzed by western blot using antibodies against ERK and pERK, with α-tubulin for normalization (Fig. 3A). Normalized ERK levels showed no difference between wild-type and RSKΔ58/1 preparations (Fig. 3B; wild type=1.00±0.12; RSKΔ58/1=1.02±0.11), indicating that loss of RSK has no influence on overall ERK levels. By contrast, pERK levels were significantly increased in RSKΔ58/1 compared with wild type (Fig. 3C; wild type=1.00±0.14; RSKΔ58/1=1.25±0.16, P<0.001). To validate the observed changes in pERK activity, a 20 kb genomic rescue construct encompassing the 6 kb RSK transcription unit (in the following, termed P[RSK]) was introduced into a RSKΔ58/1 background. In RSKΔ58/1;P[RSK] animals, normalized pERK levels became significantly different from RSKΔ58/1 (Fig. 3A,C; 1.02±0.15, P<0.05 versus RSKΔ58/1) and returned to wild-type levels. In summary, the results are in line with previous genetic analyses, which indicated a function of RSK as a negative regulator of the ERK/MAPK signaling pathway (Kim et al., 2006).Fig. 3.

Bottom Line: Based on histochemical and electrophysiological analyses, we conclude that RSK is required for normal synaptic morphology and function.Elevated ERK activity was evident in the somata of motoneurons, whereas decreased ERK activity was observed in axons and the presynapse.Our results emphasize the importance of fine-tuning ERK activity in neuronal processes underlying higher brain functions.

View Article: PubMed Central - PubMed

Affiliation: University of Würzburg, Institute of Medical Radiation and Cell Research, Versbacherstraße 5, Würzburg D-97078, Germany.

No MeSH data available.


Related in: MedlinePlus