Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin-severing protein, plays key roles in muscle sarcomere addition and maintenance. Our previous work found that Drosophila cofilin (DmCFL) knockdown in muscle causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA-sequencing analysis that unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL muscle knockdown causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown resulted in mislocalization of GluRIIA class glutamate receptors in more deteriorated muscles and strongly impaired NMJ transmission strength. These findings expand our understanding of the roles of cofilin in muscle to include NMJ structural development and suggest that NMJ defects may contribute to the pathophysiology of nemaline myopathy.

PTPN11/Corkscrew Activates Local Presynaptic Mapk Signaling to Regulate Synapsin, Synaptic Vesicle Pools, and Neurotransmission Strength, with a Dual Requirement in Neurons and Glia.

Cytoplasmic protein tyrosine phosphatase nonreceptor type 11 (PTPN11) and Drosophila homolog Corkscrew (Csw) regulate the mitogen-activated protein kinase (MAPK) pathway via a conserved autoinhibitory mechanism. Disease-causing loss-of-function (LoF) and gain-of-function (GoF) mutations both disrupt this autoinhibition to potentiate MAPK signaling. At the Drosophila neuromuscular junction glutamatergic synapse, LoF/GoF mutations elevate transmission strength and reduce activity-dependent synaptic depression. In both sexes of LoF/GoF mutations, the synaptic vesicles (SV)-colocalized synapsin phosphoprotein tether is highly elevated at rest, but quickly reduced with stimulation, suggesting a larger SV reserve pool with greatly heightened activity-dependent recruitment. Transmission electron microscopy of mutants reveals an elevated number of SVs clustered at the presynaptic active zones, suggesting that the increased vesicle availability is causative for the elevated neurotransmission. Direct neuron-targeted extracellular signal-regulated kinase (ERK) GoF phenocopies both increased local presynaptic MAPK/ERK signaling and synaptic transmission strength in mutants, confirming the presynaptic regulatory mechanism. Synapsin loss blocks this elevation in both presynaptic PTPN11 and ERK mutants. However, csw null mutants cannot be rescued by wild-type Csw in neurons: neurotransmission is only rescued by expressing Csw in both neurons and glia simultaneously. Nevertheless, targeted LoF/GoF mutations in either neurons or glia alone recapitulate the elevated neurotransmission. Thus, PTPN11/Csw mutations in either cell type are sufficient to upregulate presynaptic function, but a dual requirement in neurons and glia is necessary for neurotransmission. Taken together, we conclude that PTPN11/Csw acts in both neurons and glia, with LoF and GoF similarly upregulating MAPK/ERK signaling to enhance presynaptic Synapsin-mediated SV trafficking.

Muscle cofilin alters neuromuscular junction postsynaptic development to strengthen functional neurotransmission.

Cofilin, an actin severing protein, plays critical roles in muscle sarcomere addition and maintenance. Our previous work has shown Drosophila cofilin (DmCFL) knockdown causes progressive deterioration of muscle structure and function and produces features seen in nemaline myopathy (NM) caused by cofilin mutations. We hypothesized that disruption of actin cytoskeleton dynamics by DmCFL knockdown would impact other aspects of muscle development, and, thus, conducted an RNA sequencing analysis which unexpectedly revealed upregulated expression of numerous neuromuscular junction (NMJ) genes. We found that DmCFL is enriched in the muscle postsynaptic compartment and that DmCFL deficiency causes F-actin disorganization in this subcellular domain prior to the sarcomere defects observed later in development. Despite NMJ gene expression changes, we found no significant changes in gross presynaptic Bruchpilot active zones or total postsynaptic glutamate receptor levels. However, DmCFL knockdown results in mislocalization of glutamate receptors containing the GluRIIA subunit in more deteriorated muscles and neurotransmission strength is strongly impaired. These findings expand our understanding of cofilin's roles in muscle to include NMJ structural development and suggest that NMJ defects may contribute to NM pathophysiology.

FMRP activity and control of Csw/SHP2 translation regulate MAPK-dependent synaptic transmission.

Noonan syndrome (NS) and NS with multiple lentigines (NSML) cognitive dysfunction are linked to SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) gain-of-function (GoF) and loss-of-function (LoF), respectively. In Drosophila disease models, we find both SHP2 mutations from human patients and corkscrew (csw) homolog LoF/GoF elevate glutamatergic transmission. Cell-targeted RNAi and neurotransmitter release analyses reveal a presynaptic requirement. Consistently, all mutants exhibit reduced synaptic depression during high-frequency stimulation. Both LoF and GoF mutants also show impaired synaptic plasticity, including reduced facilitation, augmentation, and post-tetanic potentiation. NS/NSML diseases are characterized by elevated MAPK/ERK signaling, and drugs suppressing this signaling restore normal neurotransmission in mutants. Fragile X syndrome (FXS) is likewise characterized by elevated MAPK/ERK signaling. Fragile X Mental Retardation Protein (FMRP) binds csw mRNA and neuronal Csw protein is elevated in Drosophila fragile X mental retardation 1 (dfmr1) nulls. Moreover, phosphorylated ERK (pERK) is increased in dfmr1 and csw null presynaptic boutons. We find presynaptic pERK activation in response to stimulation is reduced in dfmr1 and csw nulls. Trans-heterozygous csw/+; dfmr1/+ recapitulate elevated presynaptic pERK activation and function, showing FMRP and Csw/SHP2 act within the same signaling pathway. Thus, a FMRP and SHP2 MAPK/ERK regulative mechanism controls basal and activity-dependent neurotransmission strength.

RNA-binding FMRP and Staufen sequentially regulate the Coracle scaffold to control synaptic glutamate receptor and bouton development.

Both mRNA-binding Fragile X mental retardation protein (FMRP; Fmr1) and mRNA-binding Staufen regulate synaptic bouton formation and glutamate receptor (GluR) levels at the Drosophila neuromuscular junction (NMJ) glutamatergic synapse. Here, we tested whether these RNA-binding proteins act jointly in a common mechanism. We found that both dfmr1 and staufen mutants, and trans-heterozygous double mutants, displayed increased synaptic bouton formation and GluRIIA accumulation. With cell-targeted RNA interference, we showed a downstream Staufen role within postsynaptic muscle. With immunoprecipitation, we showed that FMRP binds staufen mRNA to stabilize postsynaptic transcripts. Staufen is known to target actin-binding, GluRIIA anchor Coracle, and we confirmed that Staufen binds to coracle mRNA. We found that FMRP and Staufen act sequentially to co-regulate postsynaptic Coracle expression, and showed that Coracle, in turn, controls GluRIIA levels and synaptic bouton development. Consistently, we found that dfmr1, staufen and coracle mutants elevate neurotransmission strength. We also identified that FMRP, Staufen and Coracle all suppress pMad activation, providing a trans-synaptic signaling linkage between postsynaptic GluRIIA levels and presynaptic bouton development. This work supports an FMRP-Staufen-Coracle-GluRIIA-pMad pathway regulating structural and functional synapse development.

Yeast synaptobrevin, Snc1, engages distinct routes of postendocytic recycling mediated by a sorting nexin, Rcy1-COPI, and retromer.

The budding yeast v-SNARE, Snc1, mediates fusion of exocytic vesicles to the plasma membrane (PM) and is subsequently recycled back to the Golgi. Postendocytic recycling of Snc1 requires a phospholipid flippase (Drs2-Cdc50), an F-box protein (Rcy1), a sorting nexin (Snx4-Atg20), and the COPI coat complex. A portion of the endocytic tracer FM4-64 is also recycled back to the PM after internalization. However, the relationship between Snx4, Drs2, Rcy1, and COPI in recycling Snc1 or FM4-64 is unclear. Here we show that rcy1∆ and drs2∆ single mutants, or a COPI mutant deficient in ubiquitin binding, display a defect in recycling FM4-64 while snx4∆ cells recycle FM4-64 normally. The addition of latrunculin A to acutely inhibit endocytosis shows that rcy1∆ and snx4∆ single mutants retain the ability to recycle Snc1, but a snx4∆rcy1∆ mutant substantially blocks export. Additional deletion of a retromer subunit completely eliminates recycling of Snc1 in the triple mutant (snx4∆rcy1∆vps35∆). A minor role for retromer in Snc1 recycling can also be observed in single and double mutants harboring vps35∆. These data support the existence of three distinct and parallel recycling pathways mediated by Drs2/Rcy1/COPI, Snx4-Atg20, and retromer that retrieve an exocytic v-SNARE from the endocytic pathway to the Golgi.

Carrier of Wingless (Cow) Regulation of Drosophila Neuromuscular Junction Development.

The first Wnt signaling ligand discovered, Drosophila Wingless [Wg (Wnt1 in mammals)], plays critical roles in neuromuscular junction (NMJ) development, regulating synaptic architecture, and function. Heparan sulfate proteoglycans (HSPGs), consisting of a core protein with heparan sulfate (HS) glycosaminoglycan (GAG) chains, bind to Wg ligands to control both extracellular distribution and intercellular signaling function. Drosophila HSPGs previously shown to regulate Wg trans-synaptic signaling at the NMJ include the glypican Dally-like protein (Dlp) and perlecan Terribly Reduced Optic Lobes (Trol). Here, we investigate synaptogenic functions of the most recently described Drosophila HSPG, secreted Carrier of Wingless (Cow), which directly binds Wg in the extracellular space. At the glutamatergic NMJ, we find that Cow secreted from the presynaptic motor neuron acts to limit synaptic architecture and neurotransmission strength. In cow null mutants, we find increased synaptic bouton number and elevated excitatory current amplitudes, phenocopying presynaptic Wg overexpression. We show cow null mutants exhibit an increased number of glutamatergic synapses and increased synaptic vesicle fusion frequency based both on GCaMP imaging and electrophysiology recording. We find that membrane-tethered Wg prevents cow null defects in NMJ development, indicating that Cow mediates secreted Wg signaling. It was shown previously that the secreted Wg deacylase Notum restricts Wg signaling at the NMJ, and we show here that Cow and Notum work through the same pathway to limit synaptic development. We conclude Cow acts cooperatively with Notum to coordinate neuromuscular synapse structural and functional differentiation via negative regulation of Wg trans-synaptic signaling within the extracellular synaptomatrix.