The impact of cytoskeletal organization on the local regulation of neuronal transport.

Neurons are akin to modern cities in that both are dependent on robust transport mechanisms. Like the best mass transit systems, trafficking in neurons must be tailored to respond to local requirements. Neurons depend on both high-speed, long-distance transport and localized dynamics to correctly deliver cargoes and to tune synaptic responses. Here, we focus on the mechanisms that provide localized regulation of the transport machinery, including the cytoskeleton and molecular motors, to yield compartment-specific trafficking in the axon initial segment, axon terminal, dendrites and spines. The synthesis of these mechanisms provides a sophisticated and responsive transit system for the cell.

Dynein efficiently navigates the dendritic cytoskeleton to drive the retrograde trafficking of BDNF/TrkB signaling endosomes.

The efficient transport of cargoes within axons and dendrites is critical for neuronal function. Although we have a basic understanding of axonal transport, much less is known about transport in dendrites. We used an optogenetic approach to recruit motor proteins to cargo in real time within axons or dendrites in hippocampal neurons. Kinesin-1, a robust axonal motor, moves cargo less efficiently in dendrites. In contrast, cytoplasmic dynein efficiently navigates both axons and dendrites; in both compartments, dynamic microtubule plus ends enhance dynein-dependent transport. To test the predictions of the optogenetic assay, we examined the contribution of dynein to the motility of an endogenous dendritic cargo and found that dynein inhibition eliminates the retrograde bias of BDNF/TrkB trafficking. However, inhibition of microtubule dynamics has no effect on BDNF/TrkB motility, suggesting that dendritic kinesin motors may cooperate with dynein to drive the transport of signaling endosomes into the soma. Collectively our data highlight compartment-specific differences in kinesin activity that likely reflect specialized tuning for localized cytoskeletal determinants, whereas dynein activity is less compartment specific but is more responsive to changes in microtubule dynamics.

Activity-Dependent Regulation of Distinct Transport and Cytoskeletal Remodeling Functions of the Dendritic Kinesin KIF21B.

The dendritic arbor is subject to continual activity-dependent remodeling, requiring a balance between directed cargo trafficking and dynamic restructuring of the underlying microtubule tracks. How cytoskeletal components are able to dynamically regulate these processes to maintain this balance remains largely unknown. By combining single-molecule assays and live imaging in rat hippocampal neurons, we have identified the kinesin-4 KIF21B as a molecular regulator of activity-dependent trafficking and microtubule dynamicity in dendrites. We find that KIF21B contributes to the retrograde trafficking of brain-derived neurotrophic factor (BDNF)-TrkB complexes and also regulates microtubule dynamics through a separable, non-motor microtubule-binding domain. Neuronal activity enhances the motility of KIF21B at the expense of its role in cytoskeletal remodeling, the first example of a kinesin whose function is directly tuned to neuronal activity state. These studies suggest a model in which KIF21B navigates the complex cytoskeletal environment of dendrites by compartmentalizing functions in an activity-dependent manner.

Lipid Rafts Assemble Dynein Ensembles.

New work by Rai et al. identifies a novel mechanism regulating phagosome transport in cells: the clustering of dynein motors into lipid microdomains, leading to enhanced unidirectional motility. Clustering may be especially important for dynein, a motor that works most efficiently in teams.

The Kinesin KIF21B Regulates Microtubule Dynamics and Is Essential for Neuronal Morphology, Synapse Function, and Learning and Memory.

The kinesin KIF21B is implicated in several human neurological disorders, including delayed cognitive development, yet it remains unclear how KIF21B dysfunction may contribute to pathology. One limitation is that relatively little is known about KIF21B-mediated physiological functions. Here, we generated Kif21b knockout mice and used cellular assays to investigate the relevance of KIF21B in neuronal and in vivo function. We show that KIF21B is a processive motor protein and identify an additional role for KIF21B in regulating microtubule dynamics. In neurons lacking KIF21B, microtubules grow more slowly and persistently, leading to tighter packing in dendrites. KIF21B-deficient neurons exhibit decreased dendritic arbor complexity and reduced spine density, which correlate with deficits in synaptic transmission. Consistent with these observations, Kif21b-null mice exhibit behavioral changes involving learning and memory deficits. Our study provides insight into the cellular function of KIF21B and the basis for cognitive decline resulting from KIF21B dysregulation.

Molecular mechanisms of activity-dependent changes in dendritic morphology: role of RGK proteins.

The nervous system has the amazing capacity to transform sensory experience from the environment into changes in neuronal activity that, in turn, cause long-lasting alterations in neuronal morphology. Recent findings indicate that, surprisingly, sensory experience concurrently activates molecular signaling pathways that both promote and inhibit dendritic complexity. Historically, a number of positive regulators of activity-dependent dendritic complexity have been described, whereas the list of identified negative regulators of this process is much shorter. In recent years, there has been an emerging appreciation of the importance of the Rad/Rem/Rem2/Gem/Kir (RGK) GTPases as mediators of activity-dependent structural plasticity. In the following review, we discuss the traditional view of RGK proteins, as well as our evolving understanding of the role of these proteins in instructing structural plasticity.

Rem2 is an activity-dependent negative regulator of dendritic complexity in vivo.

A key feature of the CNS is structural plasticity, the ability of neurons to alter their morphology and connectivity in response to sensory experience and other changes in the environment. How this structural plasticity is achieved at the molecular level is not well understood. We provide evidence that changes in sensory experience simultaneously trigger multiple signaling pathways that either promote or restrict growth of the dendritic arbor; structural plasticity is achieved through a balance of these opposing signals. Specifically, we have uncovered a novel, activity-dependent signaling pathway that restricts dendritic arborization. We demonstrate that the GTPase Rem2 is regulated at the transcriptional level by calcium influx through L-VGCCs and inhibits dendritic arborization in cultured rat cortical neurons and in the Xenopus laevis tadpole visual system. Thus, our results demonstrate that changes in neuronal activity initiate competing signaling pathways that positively and negatively regulate the growth of the dendritic arbor. It is the balance of these opposing signals that leads to proper dendritic morphology.

A loss-of-function analysis reveals that endogenous Rem2 promotes functional glutamatergic synapse formation and restricts dendritic complexity.

Rem2 is a member of the RGK family of small Ras-like GTPases whose expression and function is regulated by neuronal activity in the brain. A number of questions still remain as to the endogenous functions of Rem2 in neurons. RNAi-mediated Rem2 knockdown leads to an increase in dendritic complexity and a decrease in functional excitatory synapses, though a recent report challenged the specificity of Rem2-targeted RNAi reagents. In addition, overexpression in a number of cell types has shown that Rem2 can inhibit voltage-gated calcium channel (VGCC) function, while studies employing RNAi-mediated knockdown of Rem2 have failed to observe a corresponding enhancement of VGCC function. To further investigate these discrepancies and determine the endogenous function of Rem2, we took a comprehensive, loss-of-function approach utilizing two independent, validated Rem2-targeted shRNAs to analyze Rem2 function. We sought to investigate the consequence of endogenous Rem2 knockdown by focusing on the three reported functions of Rem2 in neurons: regulation of synapse formation, dendritic morphology, and voltage-gated calcium channels. We conclude that endogenous Rem2 is a positive regulator of functional, excitatory synapse development and a negative regulator of dendritic complexity. In addition, while we are unable to reach a definitive conclusion as to whether the regulation of VGCCs is an endogenous function of Rem2, our study reports important data regarding RNAi reagents for use in future investigation of this issue.

CaMKII-dependent phosphorylation of the GTPase Rem2 is required to restrict dendritic complexity.

The morphogenesis of the dendritic arbor is a critical aspect of neuronal development, ensuring that proper neural networks are formed. However, the molecular mechanisms that underlie this dendritic remodeling remain obscure. We previously established the activity-regulated GTPase Rem2 as a negative regulator of dendritic complexity. In this study, we identify a signaling pathway whereby Rem2 regulates dendritic arborization through interactions with Ca(2+)/calmodulin-dependent kinases (CaMKs) in rat hippocampal neurons. Specifically, we demonstrate that Rem2 functions downstream of CaMKII but upstream of CaMKIV in a pathway that restricts dendritic complexity. Furthermore, we show that Rem2 is a novel substrate of CaMKII and that phosphorylation of Rem2 by CaMKII regulates Rem2 function and subcellular localization. Overall, our results describe a unique signal transduction network through which Rem2 and CaMKs function to restrict dendritic complexity.

The GTPase Rem2 regulates synapse development and dendritic morphology.

Rem2 is a member of the Rad/Rem/Rem2/Gem/Kir subfamily of small Ras-like GTPases that was identified as an important mediator of synapse development. We performed a comprehensive, loss- of-function analysis of Rem2 function in cultured hippocampal neurons using RNAi to substantially decrease Rem2 protein levels. We found that knockdown of Rem2 decreases the density and maturity of dendritic spines, the primary site of excitatory synapses onto pyramidal neurons in the hippocampus. Knockdown of Rem2 also alters the gross morphology of dendritic arborizations, increasing the number of dendritic branches without altering total neurite length. Thus, Rem2 functions to inhibit dendritic branching and promote the development of dendritic spines and excitatory synapses. Interestingly, binding to the calcium-binding protein calmodulin is required for the Rem2 regulation of dendritic branching. However, this interaction is completely dispensable for synapse development. Overall, our results suggest that Rem2 regulates dendritic branching and synapse development via distinct and overlapping signal transduction pathways.