Prodomain Removal Enables Neto to Stabilize Glutamate Receptors at the Drosophila Neuromuscular Junction

Abstract

Stabilization of neurotransmitter receptors at postsynaptic specializations is a key step in the assembly of functional synapses. Drosophila Neto (Neuropillin and Tolloid-like protein) is an essential auxiliary subunit of ionotropic glutamate receptor (iGluR) complexes required for the iGluRs clustering at the neuromuscular junction (NMJ). Here we show that optimal levels of Neto are crucial for stabilization of iGluRs at synaptic sites and proper NMJ development. Genetic manipulations of Neto levels shifted iGluRs distribution to extrajunctional locations. Perturbations in Neto levels also produced small NMJs with reduced synaptic transmission, but only Neto-depleted NMJs showed diminished postsynaptic components. Drosophila Neto contains an inhibitory prodomain that is processed by Furin1-mediated limited proteolysis. neto null mutants rescued with a Neto variant that cannot be processed have severely impaired NMJs and reduced iGluRs synaptic clusters. Unprocessed Neto retains the ability to engage iGluRs in vivo and to form complexes with normal synaptic transmission. However, Neto prodomain must be removed to enable iGluRs synaptic stabilization and proper postsynaptic differentiation.

Ultrastructural comparison of theDrosophilalarval and adult ventral abdominal neuromuscular junction

“…Although a basic electrophysiological characterization of the adult VLM NMJ still needs to be done, I hypothesize that the smaller membrane apposition that has been found in this study is of functional importance and might be associated with different electrophysiological properties. In support of such a possibility is the observation that in neto mutants where the membrane apposition at synaptic sites was found to be smaller, there was a parallel reduction in GluRIIA levels and a reduction in the amplitude of spontaneous miniature excitatory postsynaptic potentials (mEPSPs), demonstrating that these morphological alterations correlate with a functional deficit that is consistent with altered GluRIIA abundance (Kim et al, ; Ramos et al, ). Interestingly, a similar phenotype has been described in hypomorphic gluRIIA mutants (Schmid et al, ).…”

“…A properly formed membrane apposition seems to be essential for the correct targeting and maintenance of glutamate receptors (GluRs), a prerequisite for normal synaptic transmission. Mutations in neto (Neuropillin and Tolloid‐like), coding for an obligatory auxiliary subunit of the GluR complexes, are associated with alterations of membrane apposition formation and postsynaptic GluR accumulation (Kim, Bao, Bonanno, Zhang., & Serpe, 2012; Kim et al, ; Ramos, Igiesuorobo, Wang, & Serpe, ). Although a basic electrophysiological characterization of the adult VLM NMJ still needs to be done, I hypothesize that the smaller membrane apposition that has been found in this study is of functional importance and might be associated with different electrophysiological properties.…”

“…Another morphological parameter, which was found to be altered, is the size of the SSR, being much smaller at the adult NMJ. At the larval NMJ, different postsynaptic proteins including Neto, PAK (p21‐activated kinase), and the Eps15 Homology Domain (EHD) protein Past1 are required to coordinate the formation of the SSR and that of the postsynaptic compartment including the accumulation of GluRs and different scaffolding proteins (Albin & Davis, ; Kim et al, ; Koles et al, ; Ramos et al, ). Based on the observations in Past1 and PAK mutants showing an altered SSR elaboration, reduction of GluRIIA levels, reduced mEPSPs and dampened evoked postsynaptic potentials (Albin & Davis, ; Koles et al, ), it is assumed that not only the correct membrane apposition at synaptic sites but also a properly formed SSR is necessary for normal synaptic transmission.…”

“…While five subtypes of EAATs are expressed in neurons or glial cells in the vertebrate nervous system (Danbolt, 2001;Gegelashvili & Schousboe, 1998;Seal & Amara, 1999), only one glutamate transporter, dEAAT1, is expressed in Drosophila (Besson et al, 2000;Seal et al, 1998). the postsynaptic compartment including the accumulation of GluRs and different scaffolding proteins (Albin & Davis, 2004;Kim et al, 2012Kim et al, , 2015Koles et al, 2015;Ramos et al, 2015). Based on the observations in Past1 and PAK mutants showing an altered SSR elaboration, reduction of GluRIIA levels, reduced mEPSPs and dampened evoked postsynaptic potentials (Albin & Davis, 2004;Koles et al, 2015), it is assumed that not only the correct membrane apposition at synaptic sites but also a properly formed SSR is necessary for normal synaptic transmission.…”

Neto-α controls synapse organization and homeostasis at the Drosophila neuromuscular junction

“…Indeed, Neto-β, the predominant isoform at the larval NMJ, mediates intracellular interactions that recruit PSD components and enables synaptic stabilization of selective receptor subtypes . Neto-α can rescue viability and receptor clustering defects of neto null (Kim et al, 2012;Kim et al, 2015;Ramos et al, 2015). However, the endogenous functions of Neto-α remain unknown.…”