Self-oligomerization Regulates Stability of Survival Motor Neuron Protein Isoforms by Sequestering an SCF\u3csup\u3eSlmb\u3c/sup\u3e Degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

Self-oligomerization regulates stability of survival motor neuron protein isoforms by sequestering an SCF^Slmb degron

Spinal muscular atrophy (SMA) is caused by homozygous mutations in human SMN1. Expression of a duplicate gene (SMN2) primarily results in skipping of exon 7 and production of an unstable protein isoform, SMNΔ7. Although SMN2 exon skipping is the principal contributor to SMA severity, mechanisms governing stability of survival motor neuron (SMN) isoforms are poorly understood. We used a Drosophila model system and label-free proteomics to identify the SCFSlmb ubiquitin E3 ligase complex as a novel SMN binding partner. SCFSlmb interacts with a phosphor degron embedded within the human and fruitfly SMN YG-box oligomerization domains. Substitution of a conserved serine (S270A) interferes with SCFSlmb binding and stabilizes SMNΔ7. SMA-causing missense mutations that block multimerization of full-length SMN are also stabilized in the degron mutant background. Overexpression of SMNΔ7S270A, but not wild-type (WT) SMNΔ7, provides a protective effect in SMA model mice and human motor neuron cell culture systems. Our findings support a model wherein the degron is exposed when SMN is monomeric and sequestered when SMN forms higher-order multimers

A single-cross, RNA interference-based genetic tool for examining the long-term maintenance of homeostatic plasticity

Homeostatic synaptic plasticity (HSP) helps neurons and synapses maintain physiologically appropriate levels of output. The fruit fly Drosophila melanogaster larval neuromuscular junction (NMJ) is a valuable model for studying HSP. Here we introduce a genetic tool that allows fruit fly researchers to examine the lifelong maintenance of HSP with a single cross. The tool is a fruit fly stock that combines the GAL4/UAS expression system with RNA interference (RNAi)-based knock down of a glutamate receptor subunit gene. With this stock, we uncover important new information about the maintenance of HSP. We address an open question about the role that presynaptic CaV2-type Ca2+ channels play in NMJ homeostasis. Published experiments have demonstrated that hypomorphic missense mutations in the CaV2 α1a subunit gene cacophony (cac) can impair homeostatic plasticity at the NMJ. Here we report that reducing cac expression levels by RNAi is not sufficient to impair homeostatic plasticity. The presence of wild-type channels appears to support HSP – even when total CaV2 function is severely reduced. We also conduct an RNAi- and electrophysiology-based screen to identify new factors required for sustained homeostatic signaling throughout development. We uncover novel roles in HSP for Drosophila homologs of Cysteine string protein (CSP) and Phospholipase Cβ (Plc21C). We characterize those roles through follow-up genetic tests. We discuss how CSP, Plc21C, and associated factors could modulate presynaptic CaV2 function, presynaptic Ca2+ handling, or other signaling processes crucial for sustained homeostatic regulation of NMJ function throughout development. Our findings expand the scope of signaling pathways and processes that contribute to the durable strength of the NMJ

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

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

<i>Csk</i> mutant NMJs display normal baseline neurotransmission.

<p><b>(A)</b> Values for mEPSP amplitude (black), EPSP amplitude (gray), and quantal content (QC; white) normalized to wild type (dashed line). No measures were significantly different from wild type. <b>(B)</b> Representative electrophysiological traces for the data shown in A. Scale bar for EPSP (mEPSP) traces: y = 5 mV (0.5 mV), x = 50 ms (1 s). <b>(C and D)</b> Failure analysis of wild type and <i>Csk</i> mutant NMJs. <b>(C)</b> % of total stimulus events that failed to produce an evoked response. <b>(D)</b> Quantal content calculated from failure analysis, QC = ln(# trials/# failures). <b>(E)</b> Calcium cooperativity curve from NMJs of wild type and <i>Csk</i> mutant animals. Quantal content corrected for non-linear summation (NLS) was determined at the extracellular calcium concentrations shown. <b>(F)</b> Average values for EPSP amplitudes as a percent of the initial EPSP amplitude at pulse 1, 1500, 3000, 4500, and 6000 of a 6000 pulse, 10 Hz train. <b>(G)</b> Representative electrophysiological traces for the data shown in F. Scale bar x = 1 min, y = 10 mV. For A-E, * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns—not significant (<i>p</i> > 0.1) by Student’s T-test. For (F), * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns—not significant (<i>p</i> > 0.1) by ANOVA (Tukey’s post-hoc) when comparing to wild type.</p

Excess FasII impairs synaptic homeostasis.

<p><b>(A-E)</b> Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls (dashed line) that lack a homeostatic challenge (non-<i>GluRIII KD</i> or non-<i>GluRIIA</i> controls). <b>(A)</b> Trans-synaptic FasII overexpression (O/E) from the FasII endogenous locus (<i>FasII</i>^{<i>EP1462</i>}) shows partial impairment of synaptic homeostasis, as does <b>(B)</b> muscle-specific overexpression with <i>FasII</i>^{<i>EP1462</i>}. <b>(C)</b> Overexpressing specific isoforms of FasII does not impair homeostatic compensation. <b>(D)</b> Overexpressing Csk-YFP in addition to FasII^EP1462 fails to suppress the homeostatic defects of FasII O/E seen in B. <b>(E)</b> Neither <i>FasII</i> loss-of-function mutations nor <i>FasII</i> knockdown (KD) impairs synaptic homeostasis. <b>(F)</b> Average values for synaptic FasII and Dlg fluorescence intensity normalized to synapse area. <b>(G-H)</b> Representative images of FasII immunostaining for trans-synaptic FasII overexpression. Scale bar = 10 μm. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns—not significant (<i>p</i> > 0.08) by Student’s T-test comparing homeostatically challenged mutants to their unchallenged (non-<i>GluRIIA</i>) controls.</p

Expression of a Fasciclin II isoform is lowered during synaptic homeostasis and regulated by <i>Csk</i>.

<p><b>(A-E)</b> Representative images of FasII immunostaining at NMJs that are <b>(A)</b> wild type, <b>(B)</b> <i>Csk</i>^{<i>c04256</i>}<i>/Csk</i>^<i>j1D8</i>, <b>(C)</b> expressing <i>Csk-RNAi</i> in muscle and neurons, <b>(D)</b> expressing <i>Csk-RNAi</i> in only muscle, <i>and</i> <b>(E)</b> expressing <i>Csk-RNAi</i> in only neurons. <b>(F)</b> Average values for synaptic FasII fluorescence intensity normalized to synapse area and normalized to wild type. <b>(G)</b> Western blot of FasII (DSHB 1D4 antibody) in protein extracts from whole third instar larvae. <b>(H, I)</b> Relative quantification of (H) total FasII intensity and (I) the intensity of the lowest molecular weight FasII band (the ‘third band’). Quantification in H and I is shown as a fold change relative to wild type. Values on/above bars indicate the number of biological replicates for each genotype. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001, ns—not significant (<i>p</i> > 0.2) by Student’s T-test compared to wild type.</p

Glial Csk regulates Fasciclin II localization at the NMJ.

<p><b>(A-E)</b> Immunostaining of anti-FasII (green), anti-Dlg (red), and anti-HRP (blue) at NMJs with the following genotypes: <b>(A-A”)</b> wild type, <b>(B-B”)</b> <i>Csk</i>^{<i>c04256</i>}, <b>(C-C”)</b> <i>Csk</i>^{<i>c04256</i>}<i>/Csk</i>^<i>j1D8</i>, <b>(D-D”)</b> <i>Csk-RNAi</i> expressed in the whole animal (<i>Tubulin-Gal4</i>), and <b>(E-E”)</b> <i>Csk-RNAi</i> expressed only in glia (<i>Nrv2-Gal4</i>). Extra-synaptic FasII was defined as FasII signal found outside the Dlg-stained region. Areas with high levels of extra-synaptic FasII are indicated with white arrows. Scale bar = 10 μm for A and 5 μm for A”. <b>(F)</b> Relative levels of extra-synaptic FasII debris (extra-synaptic FasII staining area/total FasII staining area) present at the synapse. Values are represented as a percent of wild type to allow for appropriate comparison between multiple immunostaining experiments. For details on quantification of extra-synaptic FasII levels, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005886#sec015" target="_blank">Methods</a>. * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 by Student’s T-test compared to wild type.</p

Csk genetically opposes Src family kinases in the context of synaptic homeostasis.

<p><b>(A-D)</b> Values for mEPSP amplitude (gray) and quantal content (QC; white) normalized to genetic controls that lack a homeostatic challenge (non-<i>GluRIIA</i> Control, dashed line). <b>(A-B)</b> Muscle-specific SFK overexpression (OE) impairs synaptic homeostasis, while neuron-specific OE does not. <b>(C)</b> <i>Src64B/+</i> mutation partially suppresses the <i>GluRIIA; Csk</i> block of synaptic homeostasis. <b>(D)</b> <i>Src42A/+</i> and <i>Src64B/+</i> genetic conditions do not confer homeostatic defects on their own. <b>(E)</b> 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). * <i>p</i> < 0.05, ** <i>p</i> < 0.01 *** <i>p</i> < 0.001 ns—not significant (p > 0.1) by Student’s T-test of homeostatically challenged mutants directly to their unchallenged (non-<i>GluRIIA</i>) controls or by ANOVA (Tukey’s post-hoc) when comparing across multiple homeostatically-challenged genotypes in a dataset.</p