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C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction.

Spring AM, Brusich DJ, Frank CA - PLoS Genet. (2016)

Bottom Line: By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII).By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals.Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America.

ABSTRACT
Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating downstream expression or localization of FasII.

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Related in: MedlinePlus

Transgenic Csk is sufficient for homeostatic compensation either presynaptically or postsynaptically.Average values for (A) mEPSP amplitude, (B) EPSP amplitude, and (C) quantal content (QC) for GAL4 driver control (concurrent elaV(C155)-Gal4, Sca-Gal4, BG57-Gal4) NMJs and those overexpressing YFP-tagged Csk on both sides of the synapse. (D-F”‘) Representative images of Csk-YFP localization at the NMJ and in the CNS, as detected by immunostaining when the construct is expressed on both sides of the synapse (D-D”), only in the muscle (E-E”), or only in neurons (F-F”‘). Neurons are marked by an anti-HRP antibody (blue) and Csk-YFP is shown in green. (D, E, and F) Images showing abdominal segments that include muscles 6/7 and images that show boutons at the NMJ. (D’, E’, and F’) Images showing a single muscle 6/7 synapse. (D”, E”, and F”) Images showing boutons from a muscle 6/7 synapse at high magnification. (D”‘, E”‘, and F”‘) Images showing the larval central nervous system. (G-I) Bouton localization of Csk-YFP (green) relative to Bruchpilot (Brp, G), the GluRIIA subunit (H), or Fasciclin II (FasII, I) at muscle 6/7 synapses at which Csk-YFP is expressed only in the muscle. (J-L) Average values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls (dashed line) that lack a homeostatic challenge (non-GluRIIA Control). * p < 0.05, ** p < 0.01, *** p < 0.001, ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants to their unchallenged (non-GluRIIA) controls and by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
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pgen.1005886.g005: Transgenic Csk is sufficient for homeostatic compensation either presynaptically or postsynaptically.Average values for (A) mEPSP amplitude, (B) EPSP amplitude, and (C) quantal content (QC) for GAL4 driver control (concurrent elaV(C155)-Gal4, Sca-Gal4, BG57-Gal4) NMJs and those overexpressing YFP-tagged Csk on both sides of the synapse. (D-F”‘) Representative images of Csk-YFP localization at the NMJ and in the CNS, as detected by immunostaining when the construct is expressed on both sides of the synapse (D-D”), only in the muscle (E-E”), or only in neurons (F-F”‘). Neurons are marked by an anti-HRP antibody (blue) and Csk-YFP is shown in green. (D, E, and F) Images showing abdominal segments that include muscles 6/7 and images that show boutons at the NMJ. (D’, E’, and F’) Images showing a single muscle 6/7 synapse. (D”, E”, and F”) Images showing boutons from a muscle 6/7 synapse at high magnification. (D”‘, E”‘, and F”‘) Images showing the larval central nervous system. (G-I) Bouton localization of Csk-YFP (green) relative to Bruchpilot (Brp, G), the GluRIIA subunit (H), or Fasciclin II (FasII, I) at muscle 6/7 synapses at which Csk-YFP is expressed only in the muscle. (J-L) Average values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls (dashed line) that lack a homeostatic challenge (non-GluRIIA Control). * p < 0.05, ** p < 0.01, *** p < 0.001, ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants to their unchallenged (non-GluRIIA) controls and by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.

Mentions: In a wild-type genetic background, concurrent muscle and nerve expression of Csk-YFP caused slight electrophysiological abnormalities, including a slight increase in quantal size (Fig 5A), but decreases in evoked postsynaptic excitation (Fig 5B) and quantal content (Fig 5C). We imaged concurrent muscle- and nerve-expressed Csk-YFP in unfixed, filleted larvae by fluorescence microscopy. We also used an anti-GFP antibody to stain fixed, filled larvae. In both cases, we observed a strong, punctate cytoplasmic Csk-YFP signal in the muscle (Fig 5D and 5D’). By immunostaining we could detect low levels of Csk-YFP clustered at the NMJ (Fig 5D’ and 5D”) and high amounts of Csk-YFP concentrated at the junction between muscles of adjacent abdominal segments. Next, we expressed Csk-YFP separately in muscles and neurons. By anti-GFP immunostaining, we saw that muscle-specific Csk-YFP protein spread throughout the muscle and clustered at the postsynaptic NMJ (Fig 5E–5E”) and muscle attachment sites (Fig 5E). By contrast, we only observed neuronally expressed Csk-YFP protein in the central nervous system (Fig 5F”). We did not observe neuronal-specific Csk-YFP protein at the presynaptic NMJ terminal (Fig 5F–5F”).


C-terminal Src Kinase Gates Homeostatic Synaptic Plasticity and Regulates Fasciclin II Expression at the Drosophila Neuromuscular Junction.

Spring AM, Brusich DJ, Frank CA - PLoS Genet. (2016)

Transgenic Csk is sufficient for homeostatic compensation either presynaptically or postsynaptically.Average values for (A) mEPSP amplitude, (B) EPSP amplitude, and (C) quantal content (QC) for GAL4 driver control (concurrent elaV(C155)-Gal4, Sca-Gal4, BG57-Gal4) NMJs and those overexpressing YFP-tagged Csk on both sides of the synapse. (D-F”‘) Representative images of Csk-YFP localization at the NMJ and in the CNS, as detected by immunostaining when the construct is expressed on both sides of the synapse (D-D”), only in the muscle (E-E”), or only in neurons (F-F”‘). Neurons are marked by an anti-HRP antibody (blue) and Csk-YFP is shown in green. (D, E, and F) Images showing abdominal segments that include muscles 6/7 and images that show boutons at the NMJ. (D’, E’, and F’) Images showing a single muscle 6/7 synapse. (D”, E”, and F”) Images showing boutons from a muscle 6/7 synapse at high magnification. (D”‘, E”‘, and F”‘) Images showing the larval central nervous system. (G-I) Bouton localization of Csk-YFP (green) relative to Bruchpilot (Brp, G), the GluRIIA subunit (H), or Fasciclin II (FasII, I) at muscle 6/7 synapses at which Csk-YFP is expressed only in the muscle. (J-L) Average values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls (dashed line) that lack a homeostatic challenge (non-GluRIIA Control). * p < 0.05, ** p < 0.01, *** p < 0.001, ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants to their unchallenged (non-GluRIIA) controls and by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4764653&req=5

pgen.1005886.g005: Transgenic Csk is sufficient for homeostatic compensation either presynaptically or postsynaptically.Average values for (A) mEPSP amplitude, (B) EPSP amplitude, and (C) quantal content (QC) for GAL4 driver control (concurrent elaV(C155)-Gal4, Sca-Gal4, BG57-Gal4) NMJs and those overexpressing YFP-tagged Csk on both sides of the synapse. (D-F”‘) Representative images of Csk-YFP localization at the NMJ and in the CNS, as detected by immunostaining when the construct is expressed on both sides of the synapse (D-D”), only in the muscle (E-E”), or only in neurons (F-F”‘). Neurons are marked by an anti-HRP antibody (blue) and Csk-YFP is shown in green. (D, E, and F) Images showing abdominal segments that include muscles 6/7 and images that show boutons at the NMJ. (D’, E’, and F’) Images showing a single muscle 6/7 synapse. (D”, E”, and F”) Images showing boutons from a muscle 6/7 synapse at high magnification. (D”‘, E”‘, and F”‘) Images showing the larval central nervous system. (G-I) Bouton localization of Csk-YFP (green) relative to Bruchpilot (Brp, G), the GluRIIA subunit (H), or Fasciclin II (FasII, I) at muscle 6/7 synapses at which Csk-YFP is expressed only in the muscle. (J-L) Average values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls (dashed line) that lack a homeostatic challenge (non-GluRIIA Control). * p < 0.05, ** p < 0.01, *** p < 0.001, ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants to their unchallenged (non-GluRIIA) controls and by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
Mentions: In a wild-type genetic background, concurrent muscle and nerve expression of Csk-YFP caused slight electrophysiological abnormalities, including a slight increase in quantal size (Fig 5A), but decreases in evoked postsynaptic excitation (Fig 5B) and quantal content (Fig 5C). We imaged concurrent muscle- and nerve-expressed Csk-YFP in unfixed, filleted larvae by fluorescence microscopy. We also used an anti-GFP antibody to stain fixed, filled larvae. In both cases, we observed a strong, punctate cytoplasmic Csk-YFP signal in the muscle (Fig 5D and 5D’). By immunostaining we could detect low levels of Csk-YFP clustered at the NMJ (Fig 5D’ and 5D”) and high amounts of Csk-YFP concentrated at the junction between muscles of adjacent abdominal segments. Next, we expressed Csk-YFP separately in muscles and neurons. By anti-GFP immunostaining, we saw that muscle-specific Csk-YFP protein spread throughout the muscle and clustered at the postsynaptic NMJ (Fig 5E–5E”) and muscle attachment sites (Fig 5E). By contrast, we only observed neuronally expressed Csk-YFP protein in the central nervous system (Fig 5F”). We did not observe neuronal-specific Csk-YFP protein at the presynaptic NMJ terminal (Fig 5F–5F”).

Bottom Line: By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII).By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals.Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, University of Iowa Carver College of Medicine, Iowa City, Iowa, United States of America.

ABSTRACT
Forms of homeostatic plasticity stabilize neuronal outputs and promote physiologically favorable synapse function. A well-studied homeostatic system operates at the Drosophila melanogaster larval neuromuscular junction (NMJ). At the NMJ, impairment of postsynaptic glutamate receptor activity is offset by a compensatory increase in presynaptic neurotransmitter release. We aim to elucidate how this process operates on a molecular level and is preserved throughout development. In this study, we identified a tyrosine kinase-driven signaling system that sustains homeostatic control of NMJ function. We identified C-terminal Src Kinase (Csk) as a potential regulator of synaptic homeostasis through an RNAi- and electrophysiology-based genetic screen. We found that Csk loss-of-function mutations impaired the sustained expression of homeostatic plasticity at the NMJ, without drastically altering synapse growth or baseline neurotransmission. Muscle-specific overexpression of Src Family Kinase (SFK) substrates that are negatively regulated by Csk also impaired NMJ homeostasis. Surprisingly, we found that transgenic Csk-YFP can support homeostatic plasticity at the NMJ when expressed either in the muscle or in the nerve. However, only muscle-expressed Csk-YFP was able to localize to NMJ structures. By immunostaining, we found that Csk mutant NMJs had dysregulated expression of the Neural Cell Adhesion Molecule homolog Fasciclin II (FasII). By immunoblotting, we found that levels of a specific isoform of FasII were decreased in homeostatically challenged GluRIIA mutant animals-but markedly increased in Csk mutant animals. Additionally, we found that postsynaptic overexpression of FasII from its endogenous locus was sufficient to impair synaptic homeostasis, and genetically reducing FasII levels in Csk mutants fully restored synaptic homeostasis. Based on these data, we propose that Csk and its SFK substrates impinge upon homeostatic control of NMJ function by regulating downstream expression or localization of FasII.

Show MeSH
Related in: MedlinePlus