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.

The means to specifically ablate cells inside of a living organism have recently been improved and facilitated by stable mouse lines, carrying conditional expression constructs for diphtheria toxin (DT) or diphtheria toxin receptor, that could be activated upon Cre-mediated recombination or the application of diphtheria toxin, respectively. We have lately described the R26:lacZ/DT-A line (Brockschnieder et al., 2004, Mol Cell Biol 24:7636-7642) in which a loxP-conditional DTA allele was introduced into the ubiquitously expressed Rosa26 locus. This strain allowed the ablation of a wide spectrum of cell types by crossing it to tissue specific Cre lines. Unexpectedly, homozygous (but not heterozygous) animals of the R26:lacZ/DT-A line developed some degenerative abnormalities in a variety of tissues. The defects were most probably caused by leaky expression of small amounts of toxin from the unrecombined lacZ(flox)DT-A cassette. Here we show that insertion of an additional transcriptional regulatory sequence (bovine growth hormone polyadenylation signal, bpA) following the lacZ open reading frame prevented the formation of any defects in homozygous mice. The modification did not affect the functionality of the lacZ(flox)DTA allele, as exemplified by the complete ablation of oligodendrocytes upon Cre-mediated recombination. The novel R26:lacZbpA(flox)DTA line is expected to greatly facilitate the reliable generation of cell type ablated mice.

Significance Synapses form and change in response to neuronal activity, and they dynamically exchange transmembrane proteins over time. Most synaptic proteins are synthesized in the cell body and undergo long-distance vesicular transport powered by molecular motors along microtubules. Here we show that two synaptic key proteins (GluA2 and N-cadherin) are simultaneously delivered within distinct transport vesicles through motor proteins. Our data suggest that multidomain cargo adaptors tether synaptic proteins destined for the same subcellular compartment. We propose that vesicular presorting is an alternative mechanism to efficiently supply synapses.

Neuroligins are postsynaptic cell adhesion molecules that associate with presynaptic neurexins. Both factors form a transsynaptic connection, mediate signalling across the synapse, specify synaptic functions and play a role in synapse formation. Neuroligin dysfunction impairs synaptic transmission, disrupts neuronal networks and is thought to participate in cognitive diseases. Here we report that chemical treatment designed to induce LTP or LTD induces neuroligin 1/3 turnover, leading to either increased or decreased surface membrane protein levels, respectively. Despite its structural role at a crucial transsynaptic position, GFP-neuroligin 1 leaves synapses in hippocampal neurons over time with chemical LTD-induced neuroligin internalization depending on an intact microtubule cytoskeleton. Accordingly, neuroligin 1 and its binding partner PSD-95 associate with components of the dynein motor complex and undergo retrograde co-transport with a dynein-subunit. Transgenic depletion of dynein function in mice causes postsynaptic NLG1/3 and PSD-95 enrichment. In parallel, postsynaptic density (PSD) lengths and spine head sizes are significantly increased, a similar phenotype as observed upon transgenic overexpression of NLG1 (Dahlhaus et al., 2009). Moreover, application of a competitive PSD-95 peptide or neuroligin 1 C-terminal mutagenesis, specifically alter neuroligin 1 surface membrane expression and interfere with its internalization. Our data suggest the concept that synaptic plasticity regulates neuroligin turnover through active cytoskeleton transport.

Cellular prion protein (PrP) modulates cell adhesion and signaling in the brain. Conversion to its infectious isoform causes neurodegeneration, including Creutzfeldt-Jakob disease in humans. PrP undergoes rapid plasma membrane turnover and extracellular release via exosomes. However, the intracellular transport of PrP and its potential impact on prion disease progression is barely understood. Here we identify critical components of PrP trafficking that also link intracellular and extracellular PrP turnover. PrP associates with muskelin, dynein, and KIF5C at transport vesicles. Notably, muskelin coordinates bidirectional PrP transport and facilitates lysosomal degradation over exosomal PrP release. Muskelin gene knockout consequently causes PrP accumulation at the neuronal surface and on secreted exosomes. Moreover, prion disease onset is accelerated following injection of pathogenic prions into muskelin knockout mice. Our data identify an essential checkpoint in PrP turnover. They propose a novel connection between neuronal intracellular lysosome targeting and extracellular exosome trafficking, relevant to the pathogenesis of neurodegenerative conditions.