Membrane‐bounded organelles (MBOs) are delivered to different domains in neurons by fast axonal transport. The importance of kinesin for fast antero grade transport is well established, but mechanisms for regulating kinesin‐based motility are largely unknown. In this report, we provide biochemical and in vivo evidence that kinesin light chains (KLCs) interact with and are in vivo substrates for glycogen synthase kinase 3 (GSK3). Active GSK3 inhibited anterograde, but not retrograde, transport in squid axoplasm and reduced the amount of kinesin bound to MBOs. Kinesin microtubule binding and microtubule‐stimulated ATPase activities were unaffected by GSK3 phosphorylation of KLCs. Active GSK3 was also localized preferentially to regions known to be sites of membrane delivery. These data suggest that GSK3 can regulate fast anterograde axonal transport and targeting of cargos to specific subcellular domains in neurons.

No abstract

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane‐bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule‐dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin‐driven motility in axons. This pathway involves enzymatic activities of cyclin‐dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase‐3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.

Local changes in microtubule organization and distribution are required for the axon to grow and navigate appropriately; however, little is known about how microtubules (MTs) reorganize during directed axon outgrowth. We have used time-lapse digital imaging of developing cortical neurons microinjected with fluorescently labeled tubulin to follow the movements of individual MTs in two regions of the axon where directed growth occurs: the terminal growth cone and the developing interstitial branch. In both regions, transitions from quiescent to growth states were accompanied by reorganization of MTs from looped or bundled arrays to dispersed arrays and fragmentation of long MTs into short MTs. We also found that long-term redistribution of MTs accompanied the withdrawal of some axonal processes and the growth and stabilization of others. Individual MTs moved independently in both anterograde and retrograde directions to explore developing processes. Their velocities were inversely proportional to their lengths. Our results demonstrate directly that MTs move within axonal growth cones and developing interstitial branches. Our findings also provide the first direct evidence that similar reorganization and movement of individual MTs occur in the two regions of the axon where directed outgrowth occurs. These results suggest a model whereby short exploratory MTs could direct axonal growth cones and interstitial branches toward appropriate locations. Key words: microtubule; interstitial axon branch; growth cone; time-lapse fluorescent microscopy; cortical neuronal culture; cortical development; axon outgrowthThe growing axon contains a dense array of microtubules (MTs) that are individually short relative to the length of the axon but are tightly coalesced into a continuous bundle. MTs are essential for architectural support and also act as railways for the transport of various materials along the length of the axon. During growth and navigation of the axon, the MT array within the growth cone reorganizes and reorients toward the f uture direction of axon outgrowth (Sabry et al

Expansion of the polyglutamine (polyQ) stretch in the androgen receptor (AR) protein leads to spinal and bulbar muscular atrophy (SBMA), a neurodegenerative disease characterized by lower motor neuron degeneration. The pathogenic mechanisms underlying SBMA remain unknown, but recent experiments show that inhibition of fast axonal transport (FAT) by polyQ-expanded proteins, including polyQ-AR, represents a new cytoplasmic pathogenic lesion. Using pharmacological, biochemical and cell biological experiments, we found a new pathogenic pathway that is affected in SBMA and results in compromised FAT. PolyQ-AR inhibits FAT in a human cell line and in squid axoplasm through a pathway that involves activation of cJun N-terminal kinase (JNK) activity. Active JNK phosphorylated kinesin-1 heavy chains and inhibited kinesin-1 microtubule-binding activity. JNK inhibitors prevented polyQ-AR-mediated inhibition of FAT and reversed suppression of neurite formation by polyQ-AR. We propose that JNK represents a promising target for therapeutic interventions in SBMA.

Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.

Interstitial branches arise from the axon shaft, sometimes at great distances behind the primary growth cone. After a waiting period that can last for days after extension of the primary growth cone past the target, branches elongate toward their targets. Delayed interstitial branching is an important but little understood mechanism for target innervation in the developing CNS of vertebrates. One possible mechanism of collateral branch formation is that the axon shaft responds to target-derived signals independent of the primary growth cone. Another possibility is that the primary growth cone recognizes the target and demarcates specific regions of the axon for future branching. To address whether behaviors of the primary growth cone and development of interstitial branches are related, we performed high-resolution time-lapse imaging on dissociated sensorimotor cortical neurons that branch interstitially in vivo. Imaging of entire cortical neurons for periods of days revealed that the primary growth cone pauses in regions in which axon branches later develop. Pausing behaviors involve repeated cycles of collapse, retraction, and extension during which growth cones enlarge and reorganize. Remnants of reorganized growth cones are left behind on the axon shaft as active filopodial or lamellar protrusions, and axon branches subsequently emerge from these active regions of the axon shaft. In this study we propose a new model to account for target innervation in vivo by interstitial branching. Our model suggests that delayed interstitial branching results directly from target recognition by the primary growth cone.

Interstitial branching is an important mechanism for target innervation in the developing CNS. A previous study of cortical neurons in vitro showed that the terminal growth cone pauses and enlarges in regions from which interstitial axon branches later develop (Szebenyi et al., 1998). In the present study, we investigated how target-derived signals affect the morphology and behaviors of growth cones leading to development of axon branches. We used bath and local application of a targetderived growth factor, FGF-2, on embryonic pyramidal neurons from the sensorimotor cortex and used time-lapse digital imaging to monitor effects of FGF-2 on axon branching. Observations of developing neurons over periods of several days showed that bath-applied FGF-2 significantly increased growth cone size and slowed growth cone advance, leading to a threefold increase in axon branching. FGF-2 also had acute effects on growth cone morphology, promoting rapid growth of filopodia within minutes. Application of FGF-2-coated beads promoted local axon branching in close proximity to the beads. Branching was more likely to occur when the FGF-2 bead was on or near the growth cone, suggesting that distal regions of the axon are more responsive to FGF-2 than other regions of the axon shaft. Together, these results show that interstitial axon branches can be induced locally through the action of a targetderived growth factor that preferentially exerts effects on the growth cone. We suggest that, in target regions, growth factors such as FGF-2 and other branching factors may induce formation of collateral axon branches by enhancing the pausing and enlargement of primary growth cones that determine future branch points.