Tubb3 expression levels are sensitive to neuronal activity changes and determine microtubule growth and kinesin-mediated transport.

Microtubules are dynamic polymers of α/ß-tubulin. They regulate cell structure, cell division, cell migration, and intracellular transport. However, functional contributions of individual tubulin isotypes are incompletely understood. The neuron-specific ß-tubulin Tubb3 displays highest expression around early postnatal periods characterized by exuberant synaptogenesis. Although Tubb3 mutations are associated with neuronal disease, including abnormal inhibitory transmission and seizure activity in patients, molecular consequences of altered Tubb3 levels are largely unknown. Likewise, it is unclear whether neuronal activity triggers Tubb3 expression changes in neurons. In this study, we initially asked whether chemical protocols to induce long-term potentiation (cLTP) affect microtubule growth and the expression of individual tubulin isotypes. We found that growing microtubules and Tubb3 expression are sensitive to changes in neuronal activity and asked for consequences of Tubb3 downregulation in neurons. Our data revealed that reduced Tubb3 levels accelerated microtubule growth in axons and dendrites. Remarkably, Tubb3 knockdown induced a specific upregulation of Tubb4 gene expression, without changing other tubulin isotypes. We further found that Tubb3 downregulation reduces tubulin polyglutamylation, increases KIF5C motility and boosts the transport of its synaptic cargo N-Cadherin, which is known to regulate synaptogenesis and long-term potentiation. Due to the large number of tubulin isotypes, we developed and applied a computational model based on a Monte Carlo simulation to understand consequences of tubulin expression changes in silico. Together, our data suggest a feedback mechanism with neuronal activity regulating tubulin expression and consequently microtubule dynamics underlying the delivery of synaptic cargoes.

Muskelin regulates actin-dependent synaptic changes and intrinsic brain activity relevant to behavioral and cognitive processes.

Muskelin (Mkln1) is implicated in neuronal function, regulating plasma membrane receptor trafficking. However, its influence on intrinsic brain activity and corresponding behavioral processes remains unclear. Here we show that murine Mkln1 knockout causes non-habituating locomotor activity, increased exploratory drive, and decreased locomotor response to amphetamine. Muskelin deficiency impairs social novelty detection while promoting the retention of spatial reference memory and fear extinction recall. This is strongly mirrored in either weaker or stronger resting-state functional connectivity between critical circuits mediating locomotor exploration and cognition. We show that Mkln1 deletion alters dendrite branching and spine structure, coinciding with enhanced AMPAR-mediated synaptic transmission but selective impairment in synaptic potentiation maintenance. We identify muskelin at excitatory synapses and highlight its role in regulating dendritic spine actin stability. Our findings point to aberrant spine actin modulation and changes in glutamatergic synaptic function as critical mechanisms that contribute to the neurobehavioral phenotype arising from Mkln1 ablation.

A kinesin 1-protrudin complex mediates AMPA receptor synaptic removal during long-term depression.

The synaptic removal of AMPA-type glutamate receptors (AMPARs) is a core mechanism for hippocampal long-term depression (LTD). In this study, we address the role of microtubule-dependent transport of AMPARs as a driver for vesicular trafficking and sorting during LTD. Here, we show that the kinesin-1 motor KIF5A/C is strictly required for LTD expression in CA3-to-CA1 hippocampal synapses. Specifically, we find that KIF5 is required for an efficient internalization of AMPARs after NMDA receptor activation. We show that the KIF5/AMPAR complex is assembled in an activity-dependent manner and associates with microsomal membranes upon LTD induction. This interaction is facilitated by the vesicular adaptor protrudin, which is also required for LTD expression. We propose that protrudin links KIF5-dependent transport to endosomal sorting, preventing AMPAR recycling to synapses after LTD induction. Therefore, this work identifies an activity-dependent molecular motor and the vesicular adaptor protein that executes AMPAR synaptic removal during LTD.

Dynein and muskelin control myosin VI delivery towards the neuronal nucleus.

Protein transport toward the nucleus is important for translating molecular signals into gene expression changes. Interestingly, the unconventional motor protein myosin VI regulates RNA polymerase II-dependent gene transcription. Whether actin-filament-dependent myosins are actively transported to nuclear compartments remains unknown. Here, we report that neurons also contain myosin VI inside their nucleus. Notably, nuclear appearance of this actin-dependent motor depends on functional cytoplasmic dynein, a minus end-directed microtubule motor. We find that the trafficking factor muskelin assists in the formation of dynein-myosin VI interactions and further localizes to nuclear foci, enriched in the myosin. Impairment of dynein, but not myosin VI function, reduces nuclear muskelin levels. In turn, muskelin represents a critical determinant in regulating myosin VI nuclear targeting. Our data reveal that minus end-directed microtubule transport determines myosin VI subcellular localization. They suggest a pathway of cytoplasm-to-nucleus trafficking that requires muskelin and is based on dynein-myosin cross talk.

Spastin depletion increases tubulin polyglutamylation and impairs kinesin-mediated neuronal transport, leading to working and associative memory deficits.

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.

Myosin VI Drives Clathrin-Mediated AMPA Receptor Endocytosis to Facilitate Cerebellar Long-Term Depression.

Myosin VI is an actin-based cytoskeletal motor implicated in various steps of membrane trafficking. Here, we investigated whether this myosin is crucial for synaptic function and plasticity in neurons. We find that myosin VI localizes at cerebellar parallel fiber to Purkinje cell synapses and that the myosin is indispensable for long-term depression of AMPA-receptor-mediated synaptic signal transmission at this synapse. Moreover, direct visualization of GluA2-containing AMPA receptors in Purkinje cells reveals that the myosin drives removal of AMPA receptors from the surface of dendritic spines in an activity-dependent manner. Co-immunoprecipitation and super-resolution microscopy indicate that specifically the interaction of myosin VI with the clathrin adaptor component α-adaptin is important during long-term depression. Together, these data suggest that myosin VI directly promotes clathrin-mediated endocytosis of AMPA receptors in Purkinje cells to mediate cerebellar long-term depression. Our results provide insights into myosin VI function and the molecular mechanisms underlying synaptic plasticity.

Muskelin Coordinates PrPC Lysosome versus Exosome Targeting and Impacts Prion Disease Progression.

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

Radixin regulates synaptic GABAA receptor density and is essential for reversal learning and short-term memory.

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.

The LisH motif of muskelin is crucial for oligomerization and governs intracellular localization.

Neurons regulate the number of surface receptors by balancing the transport to and from the plasma membrane to adjust their signaling properties. The protein muskelin was recently identified as a key factor guiding the transport of α1 subunit-containing GABAA receptors. Here we present the crystal structure of muskelin, comprising its N-terminal discoidin domain and Lis1-homology (LisH) motif. The molecule crystallized as a dimer with the LisH motif exclusively mediating oligomerization. Our subsequent biochemical analyses confirmed that the LisH motif acts as a dimerization element in muskelin. Together with an intermolecular head-to-tail interaction, the LisH-dependent dimerization is required to assemble a muskelin tetramer. Intriguingly, our cellular studies revealed that the loss of this dimerization results in a complete redistribution of muskelin from the cytoplasm to the nucleus and impairs muskelin's function in GABAA receptor transport. These studies demonstrate that the LisH-dependent dimerization is a crucial factor for muskelin function.

GRIP1 interlinks N-cadherin and AMPA receptors at vesicles to promote combined cargo transport into dendrites.

The GluA2 subunit of AMPA-type glutamate receptors (AMPARs) regulates excitatory synaptic transmission in neurons. In addition, the transsynaptic cell adhesion molecule N-cadherin controls excitatory synapse function and stabilizes dendritic spine structures. At postsynaptic membranes, GluA2 physically binds N-cadherin, underlying spine growth and synaptic modulation. We report that N-cadherin binds to PSD-95/SAP90/DLG/ZO-1 (PDZ) domain 2 of the glutamate receptor interacting protein 1 (GRIP1) through its intracellular C terminus. N-cadherin and GluA2-containing AMPARs are presorted to identical transport vesicles for dendrite delivery, and live imaging reveals cotransport of both proteins. The kinesin KIF5 powers GluA2/N-cadherin codelivery by using GRIP1 as a multilink interface. Notably, GluA2 and N-cadherin use different PDZ domains on GRIP1 to simultaneously bind the transport complex, and interference with either binding motif impairs the turnover of both synaptic cargoes. Depolymerization of microtubules, deletion of the KIF5 motor domain, or specific blockade of AMPAR exocytosis affects delivery of GluA2/N-cadherin vesicles. At the functional level, interference with this cotransport reduces the number of spine protrusions and excitatory synapses. Our data suggest the concept that the multi-PDZ-domain adaptor protein GRIP1 can act as a scaffold at trafficking vesicles in the combined delivery of AMPARs and N-cadherin into dendrites.

Muskelin regulates actin filament- and microtubule-based GABA(A) receptor transport in neurons.

Intracellular transport regulates protein turnover including endocytosis. Because of the spatial segregation of F-actin and microtubules, internalized cargo vesicles need to employ 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 GABA(A) receptor (GABA(A)R) trafficking. GABA(A)Rs regulate synaptic transmission, plasticity, and network oscillations. GABA(A)R α1 and muskelin interact directly, undergo neuronal cotransport, and associate with myosin VI or dynein motor complexes in subsequent steps of GABA(A)R 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.

Synaptic activation modifies microtubules underlying transport of postsynaptic cargo.

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.