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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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Csk genetically opposes Src family kinases in the context of synaptic homeostasis.(A-D) Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls that lack a homeostatic challenge (non-GluRIIA Control, dashed line). (A-B) Muscle-specific SFK overexpression (OE) impairs synaptic homeostasis, while neuron-specific OE does not. (C)Src64B/+ mutation partially suppresses the GluRIIA; Csk block of synaptic homeostasis. (D)Src42A/+ and Src64B/+ genetic conditions do not confer homeostatic defects on their own. (E) Representative electrophysiological traces for data shown in C. Scale bar for EPSP (mEPSP) traces: y = 5 mV (0.5 mV), x = 50 ms (1 s). * p < 0.05, ** p < 0.01 *** p < 0.001 ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants directly to their unchallenged (non-GluRIIA) controls or by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
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pgen.1005886.g004: Csk genetically opposes Src family kinases in the context of synaptic homeostasis.(A-D) Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls that lack a homeostatic challenge (non-GluRIIA Control, dashed line). (A-B) Muscle-specific SFK overexpression (OE) impairs synaptic homeostasis, while neuron-specific OE does not. (C)Src64B/+ mutation partially suppresses the GluRIIA; Csk block of synaptic homeostasis. (D)Src42A/+ and Src64B/+ genetic conditions do not confer homeostatic defects on their own. (E) Representative electrophysiological traces for data shown in C. Scale bar for EPSP (mEPSP) traces: y = 5 mV (0.5 mV), x = 50 ms (1 s). * p < 0.05, ** p < 0.01 *** p < 0.001 ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants directly to their unchallenged (non-GluRIIA) controls or by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.

Mentions: Expression of wild-type UAS-Src64BUY1332 in the muscle completely blocked homeostatic compensation (Fig 4A). By contrast, neuronal expression of wild-type UAS-Src64BUY1332 had no effect on the NMJ’s homeostatic capacity (Fig 4A). The effects of Src42A overexpression were similar to Src64B. Driving either the wild-type UAS-Src42AWT transgene or the constitutively active UAS-Src42AYF transgene in the muscle impaired synaptic homeostasis (Fig 4B). By contrast, neuronal expression of UAS-Src42AYF had no inhibitory effect (Fig 4B). Our results are consistent with the possibility that a muscle-specific Csk/SFK regulatory interaction gates synaptic homeostasis. One qualification is that SFK overexpression had to be limited in order to circumvent embryonic and early larval lethality (see Materials and Methods and S1 Table for exact conditions for each SFK transgene). As a result, this experimental maneuver may have occluded potential Csk-SFK regulatory effects in neurons.


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)

Csk genetically opposes Src family kinases in the context of synaptic homeostasis.(A-D) Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls that lack a homeostatic challenge (non-GluRIIA Control, dashed line). (A-B) Muscle-specific SFK overexpression (OE) impairs synaptic homeostasis, while neuron-specific OE does not. (C)Src64B/+ mutation partially suppresses the GluRIIA; Csk block of synaptic homeostasis. (D)Src42A/+ and Src64B/+ genetic conditions do not confer homeostatic defects on their own. (E) Representative electrophysiological traces for data shown in C. Scale bar for EPSP (mEPSP) traces: y = 5 mV (0.5 mV), x = 50 ms (1 s). * p < 0.05, ** p < 0.01 *** p < 0.001 ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants directly to their unchallenged (non-GluRIIA) controls or by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764653&req=5

pgen.1005886.g004: Csk genetically opposes Src family kinases in the context of synaptic homeostasis.(A-D) Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls that lack a homeostatic challenge (non-GluRIIA Control, dashed line). (A-B) Muscle-specific SFK overexpression (OE) impairs synaptic homeostasis, while neuron-specific OE does not. (C)Src64B/+ mutation partially suppresses the GluRIIA; Csk block of synaptic homeostasis. (D)Src42A/+ and Src64B/+ genetic conditions do not confer homeostatic defects on their own. (E) Representative electrophysiological traces for data shown in C. Scale bar for EPSP (mEPSP) traces: y = 5 mV (0.5 mV), x = 50 ms (1 s). * p < 0.05, ** p < 0.01 *** p < 0.001 ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants directly to their unchallenged (non-GluRIIA) controls or by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.
Mentions: Expression of wild-type UAS-Src64BUY1332 in the muscle completely blocked homeostatic compensation (Fig 4A). By contrast, neuronal expression of wild-type UAS-Src64BUY1332 had no effect on the NMJ’s homeostatic capacity (Fig 4A). The effects of Src42A overexpression were similar to Src64B. Driving either the wild-type UAS-Src42AWT transgene or the constitutively active UAS-Src42AYF transgene in the muscle impaired synaptic homeostasis (Fig 4B). By contrast, neuronal expression of UAS-Src42AYF had no inhibitory effect (Fig 4B). Our results are consistent with the possibility that a muscle-specific Csk/SFK regulatory interaction gates synaptic homeostasis. One qualification is that SFK overexpression had to be limited in order to circumvent embryonic and early larval lethality (see Materials and Methods and S1 Table for exact conditions for each SFK transgene). As a result, this experimental maneuver may have occluded potential Csk-SFK regulatory effects in neurons.

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