Summary The microtubule motors kinesin and dynein function collectively to drive vesicular transport. High resolution tracking of vesicle motility in the cell indicates that transport is often bidirectional, characterized by frequent directional changes. However, the mechanisms coordinating the collective activities of oppositely-oriented motors bound to the same cargo are not well understood. To examine motor coordination, we purified neuronal transport vesicles and analyzed their motility using automated particle tracking with nanometer resolution. The motility of purified vesicles reconstituted in vitro closely models the movement of Lysotracker-positive vesicles in primary neurons, where processive bidirectional motility is interrupted with frequent directional switches, diffusional movement and pauses. Quantitative analysis indicates that vesicles co-purify with a low number of stably-bound motors: 1–5 dynein and 1–4 kinesin motors. These observations compare well to predictions from a stochastic tug-of-war model, where transport is driven by the force-dependent kinetics of teams of opposing motors in the absence of external regulation. Together, these observations indicate that vesicles move robustly with a small complement of tightly-bound motors, and suggest an efficient regulatory scheme for bidirectional motility where small changes in the number of engaged motors manifest in large changes in the motility of cargo.

How are phosphorylated kinases transported over long intracellular distances, such as in the case of axon to cell body signaling after nerve injury? Here, we show that the MAP kinases Erk1 and Erk2 are phosphorylated in sciatic nerve axoplasm upon nerve injury, concomitantly with the production of soluble forms of the intermediate filament vimentin by local translation and calpain cleavage in axoplasm. Vimentin binds phosphorylated Erks (pErk), thus linking pErk to the dynein retrograde motor via direct binding of vimentin to importin beta. Injury-induced Elk1 activation and neuronal regeneration are inhibited or delayed in dorsal root ganglion neurons from vimentin null mice, and in rats treated with a MEK inhibitor or with a peptide that prevents pErk-vimentin binding. Thus, soluble vimentin enables spatial translocation of pErk by importins and dynein in lesioned nerve.

Axoplasmic proteins containing nuclear localization signals (NLS) signal retrogradely by an unknown mechanism in injured nerve. Here we demonstrate that the importin/karyopherin alpha and beta families underlie this process. We show that importins are found in axons at significant distances from the cell body and that importin beta protein is increased after nerve lesion by local translation of axonal mRNA. This leads to formation of a high-affinity NLS binding complex that traffics retrogradely with the motor protein dynein. Trituration of synthetic NLS peptide at the injury site of axotomized dorsal root ganglion (DRG) neurons delays their regenerative outgrowth, and NLS introduction to sciatic nerve concomitantly with a crush injury suppresses the conditioning lesion induced transition from arborizing to elongating growth in L4/L5 DRG neurons. These data suggest a model whereby lesion-induced upregulation of axonal importin beta may enable retrograde transport of signals that modulate the regeneration of injured neurons.

Active transport along the axon is critical to the neuron. Motor-driven transport supplies the distal synapse with newly synthesized proteins and lipids, and clears damaged or misfolded proteins. Microtubule motors also drive long-distance signaling along the axon via signaling endosomes. While positive signaling initiated by neurotrophic factors has been well-studied, recent research has focused on stress signaling along the axon. Here, the connections between axonal transport alterations and neurodegeneration are discussed, including evidence for defective transport of vesicles, mitochondria, degradative organelles, and signaling endosomes in models of Amyotrophic Lateral Sclerosis, Huntington's, Parkinson's and Alzheimer's disease. Defects in transport are sufficient to induce neurodegeneration, but recent progress suggests that changes in retrograde signaling pathways correlate with rapidly progressive neuronal cell death. Active axonal transport maintains extended neuronal processesThe unique morphology of neurons, highly polarized cells with extended axons and dendrites, makes them particularly dependent on active intracellular transport. The transport of proteins, RNA, and organelles over long distances requires molecular motors that operate along the cellular cytoskeleton (Glossary).Two major roles for axonal transport are supply/clearance and long-distance signaling. Supply of newly synthesized proteins and lipids to the distal synapse maintains axonal activity, while misfolded and aggregated proteins are cleared from the axon by transport to the cell soma for efficient degradation [1]. Active transport of mitochondria also supplies local energy needs [2]. The second major role for active transport is the communication of intracellular signals from the distal axon to the soma, allowing the neuron to respond to changes in environment. While defects in either supply or clearance can readily be predicted to be deleterious to the health of the neuron, there has been a growing appreciation that the propagation of stress signaling along the axon may be a key neurodegenerative pathway leading to cell death [3,4].Here, we focus on recent progress linking defects in fast axonal transport to the pathogenesis of neurodegenerative diseases (reviewed in Table I). Observations from cellular and animal models have provided evidence for multiple alterations in axonal transport, including impaired organelle motility, defects in degradative pathways, impaired neurotrophic signaling, and elevated stress signaling. While it is likely that several cellular pathways may contribute to distal degeneration, recent progress suggests that changes in the balance of signaling along the

Retrograde axonal transport of cellular signals driven by dynein is vital for neuronal survival. Mouse models with defects in the retrograde transport machinery including the Loa mouse (point mutation in dynein) and the Tgdynamitin mouse (overexpression of dynamitin) exhibit mild neurodegenerative disease. Transport defects have also been observed in more rapidly progressive neurodegeneration, such as that observed in the SOD1G93A transgenic mouse model for familial ALS. Here we test the hypothesis that alterations in retrograde signaling lead to neurodegeneration. In-vivo, in-vitro and live cell imaging motility assays show mis-regulation of transport and inhibition of retrograde signaling in the SOD1G93A model. However, similar inhibition is also seen in the Loa and Tgdynamitin mouse models. Thus, slowing of retrograde signaling leads only to mild degeneration and cannot explain ALS etiology. To further pursue this question, we used a proteomics approach to investigate dynein-associated retrograde signaling. These data indicate a significant decrease in retrograde survival factors including P-Trk and P-Erk1/2, and an increase in retrograde stress factor signaling, including P-JNK, Caspase-8 and p75NTR cleavage fragment in the SOD1G93A model; similar changes are not seen in the Loa mouse. Co-cultures of motor neurons and glia expressing mutant SOD1 (mSOD1) in compartmentalized chambers indicate that inhibition of retrograde stress signaling is sufficient to block activation of cellular stress pathways and to rescue motor neurons from mSOD1-induced toxicity. Hence, a shift from survival-promoting to death-promoting retrograde signaling may be key to the rapid onset of neurodegeneration seen in ALS.