Postsynaptic long-term potentiation of inhibition (iLTP) can rely on increased GABAA receptors (GABAARs) at synapses by promoted exocytosis. However, the molecular mechanisms that enhance the clustering of postsynaptic GABAARs during iLTP remain obscure. Here we demonstrate that during chemically induced iLTP (chem-iLTP), GABAARs are immobilized and confined at synapses, as revealed by single-particle tracking of individual GABAARs in cultured hippocampal neurons. Chem-iLTP expression requires synaptic recruitment of the scaffold protein gephyrin from extrasynaptic areas, which in turn is promoted by CaMKII-dependent phosphorylation of GABAAR-β3-Ser383. Impairment of gephyrin assembly prevents chem-iLTP and, in parallel, blocks the accumulation and immobilization of GABAARs at synapses. Importantly, an increase of gephyrin and GABAAR similar to those observed during chem-iLTP in cultures were found in the rat visual cortex following an experience-dependent plasticity protocol that potentiates inhibitory transmission in vivo. Thus, phospho-GABAAR-β3-dependent accumulation of gephyrin at synapses and receptor immobilization are crucial for iLTP expression and are likely to modulate network excitability.

Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell‐autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1‐deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell‐autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.

SummaryDevelopmental axon remodeling is characterized by the selective removal of branches from axon arbors. The mechanisms that underlie such branch loss are largely unknown. Additionally, how neuronal resources are specifically assigned to the branches of remodeling arbors is not understood. Here we show that axon branch loss at the developing mouse neuromuscular junction is mediated by branch-specific microtubule severing, which results in local disassembly of the microtubule cytoskeleton and loss of axonal transport in branches that will subsequently dismantle. Accordingly, pharmacological microtubule stabilization delays neuromuscular synapse elimination. This branch-specific disassembly of the cytoskeleton appears to be mediated by the microtubule-severing enzyme spastin, which is dysfunctional in some forms of upper motor neuron disease. Our results demonstrate a physiological role for a neurodegeneration-associated modulator of the cytoskeleton, reveal unexpected cell biology of branch-specific axon plasticity and underscore the mechanistic similarities of axon loss in development and disease.

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery.activity-dependent ͉ neuron ͉ polyglutamylation ͉ synapse ͉ strychnine

Summary Intracellular transport regulates protein turnover including endocytosis. Because of the spatial segregation of F-actin and microtubules, internalized cargo vesicles need to employ retrograde myosin and dynein motors to traverse both cytoskeletal compartments. Factors specifying cargo delivery across both tracks remain unknown. We identified muskelin to interconnect retrograde F-actin- and microtubule-dependent GABAA receptor (GABAAR) trafficking. GABAARs regulate synaptic transmission, plasticity, and network oscillations. GABAAR α1 and muskelin interact directly, undergo neuronal cotransport, and associate with myosin VI or dynein motor complexes in subsequent steps of GABAAR endocytosis. Inhibition of either transport route selectively interferes with receptor internalization or degradation. Newly generated muskelin KO mice display depletion of both transport steps and a high-frequency ripple oscillation phenotype. A diluted coat color of muskelin KOs further suggests muskelin transport functions beyond neurons. Our data suggest the concept that specific trafficking factors help cargoes to traverse both F-actin- and microtubule compartments, thereby regulating their fate.

Neurotransmitter receptor density is a major variable in regulating synaptic strength. Receptors rapidly exchange between synapses and intracellular storage pools through endocytic recycling. In addition, lateral diffusion and confinement exchanges surface membrane receptors between synaptic and extrasynaptic sites. However, the signals that regulate this transition are currently unknown. GABAA receptors containing α5-subunits (GABAAR-α5) concentrate extrasynaptically through radixin (Rdx)-mediated anchorage at the actin cytoskeleton. Here we report a novel mechanism that regulates adjustable plasma membrane receptor pools in the control of synaptic receptor density. RhoA/ROCK signalling regulates an activity-dependent Rdx phosphorylation switch that uncouples GABAAR-α5 from its extrasynaptic anchor, thereby enriching synaptic receptor numbers. Thus, the unphosphorylated form of Rdx alters mIPSCs. Rdx gene knockout impairs reversal learning and short-term memory, and Rdx phosphorylation in wild-type mice exhibits experience-dependent changes when exposed to novel environments. Our data suggest an additional mode of synaptic plasticity, in which extrasynaptic receptor reservoirs supply synaptic GABAARs.

g-Aminobutyric acid type A and glycine receptors (GABA A Rs, GlyRs) are the major inhibitory neurotransmitter receptors and contribute to many synaptic functions, dysfunctions and human diseases. GABA A Rs are important drug targets regulated by direct interactions with the scaffolding protein gephyrin. Here we deduce the molecular basis of this interaction by chemical, biophysical and structural studies of the gephyrin-GABA A R a3 complex, revealing that the N-terminal region of the a3 peptide occupies the same binding site as the GlyR b subunit, whereas the C-terminal moiety, which is conserved among all synaptic GABA A R a subunits, engages in unique interactions. Thermodynamic dissections of the gephyrinreceptor interactions identify two residues as primary determinants for gephyrin's subunit preference. This first structural evidence for the gephyrin-mediated synaptic accumulation of GABA A Rs offers a framework for future investigations into the regulation of inhibitory synaptic strength and for the development of mechanistically and therapeutically relevant compounds targeting the gephyrin-GABA A R interaction.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.