Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain

Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

Interleukin-6 is an efficacious marker of axonal transport disruption during experimental glaucoma and stimulates neuritogenesis in cultured retinal ganglion cells

It is increasingly recognised that chronically activated glia contribute to the pathology of various neurodegenerative diseases, including glaucoma. One means by which this can occur is through the release of neurotoxic, proinflammatory factors. In the current study, we therefore investigated the spatio-temporal patterns of expression of three such cytokines, IL-1β, TNFα and IL-6, in a validated rat model of experimental glaucoma. First, only weak evidence was found for increased expression of IL-1β and TNFα following induction of ocular hypertension. Second, and much more striking, was that robust evidence was uncovered showing IL-6 to be synthesised by injured retinal ganglion cells following elevation of intraocular pressure and transported in an orthograde fashion along the nerve, accumulating at sites of axonal disruption in the optic nerve head. Verification that IL-6 represents a novel marker of disrupted axonal transport in this model was obtained by performing double labelling immunofluorescence with recognised markers of fast axonal transport. The stimulus for IL-6 synthesis and axonal transport during experimental glaucoma arose from axonal injury rather than ocular hypertension, as the response was identical after optic nerve crush and bilateral occlusion of the carotid arteries, each of which is independent of elevated intraocular pressure. Moreover, the response of IL-6 was not a generalised feature of the gp130 family of cytokines, as it was not mimicked by another family member, ciliary neurotrophic factor. Finally, further study suggested that IL-6 may be an early part of the endogenous regenerative response as the cytokine colocalised with growth-associated membrane phosphoprotein-43 in some putative regenerating axons, and potently stimulated neuritogenesis in retinal ganglion cells in culture, an effect that was additive to that of ciliary neurotrophic factor. These data comprise clear evidence that IL-6 is actively involved in the attempt of injured retinal ganglion cells to regenerate their axons.

The interaction between cytoplasmic dynein and dynactin is required for fast axonal transport

Fast axonal transport is characterized by the bidirectional, microtubule-based movement of membranous organelles. Cytoplasmic dynein is necessary but not sufficient for retrograde transport directed from the synapse to the cell body. Dynactin is a heteromultimeric protein complex, enriched in neurons, that binds to both microtubules and cytoplasmic dynein. To determine whether dynactin is required for retrograde axonal transport, we examined the effects of anti-dynactin antibodies on organelle transport in extruded axoplasm. Treatment of axoplasm with antibodies to the p150Glued subunit of dynactin resulted in a significant decrease in the velocity of microtubule-based organelle transport, with many organelles bound along microtubules. We examined the molecular mechanism of the observed inhibition of motility, and we demonstrated that antibodies to p150Glued disrupted the binding of cytoplasmic dynein to dynactin and also inhibited the association of cytoplasmic dynein with organelles. In contrast, the anti-p150Glued antibodies had no effect on the binding of dynactin to microtubules nor on cytoplasmic dynein-driven microtubule gliding. These results indicate that the interaction between cytoplasmic dynein and the dynactin complex is required for the axonal transport of membrane-bound vesicles and support the hypothesis that dynactin may function as a link between the organelle, the microtubule, and cytoplasmic dynein during vesicle transport.

Real-Time Imaging of the Axonal Transport of Granules Containing a Tissue Plasminogen Activator/Green Fluorescent Protein Hybrid

A hybrid protein, tPA/GFP, consisting of rat tissue plasminogen activator (tPA) and green fluorescent protein (GFP) was expressed in PC12 cells and used to study the distribution, secretory behavior, and dynamics of secretory granules containing tPA in living cells with a neuronal phenotype. High-resolution images demonstrate that tPA/GFP has a growth cone-biased distribution in differentiated cells and that tPA/GFP is transported in granules of the regulated secretory pathway that colocalize with granules containing secretogranin II. Time-lapse images of secretion reveal that secretagogues induce substantial loss of cellular tPA/GFP fluorescence, most importantly from growth cones. Time-lapse images of the axonal transport of granules containing tPA/GFP reveal a surprising complexity to granule dynamics. Some granules undergo canonical fast axonal transport; others move somewhat more slowly, especially in highly fluorescent neurites. Most strikingly, granules traffic bidirectionally along neurites to an extent that depends on granule accumulation, and individual granules can reverse their direction of motion. The retrograde component of this bidirectional transport may help to maintain cellular homeostasis by transporting excess tPA/GFP back toward the cell body. The results presented here provide a novel view of the axonal transport of secretory granules. In addition, the results suggest that tPA is targeted for regulated secretion from growth cones of differentiated cells, strategically positioning tPA to degrade extracellular barriers or to activate other barrier-degrading proteases during axonal elongation.

Cytoplasmic Dynein, the Dynactin Complex, and Kinesin Are Interdependent and Essential for Fast Axonal Transport

In axons, organelles move away from (anterograde) and toward (retrograde) the cell body along microtubules. Previous studies have provided compelling evidence that conventional kinesin is a major motor for anterograde fast axonal transport. It is reasonable to expect that cytoplasmic dynein is a fast retrograde motor, but relatively few tests of dynein function have been reported with neurons of intact organisms. In extruded axoplasm, antibody disruption of kinesin or the dynactin complex (a dynein activator) inhibits both retrograde and anterograde transport. We have tested the functions of the cytoplasmic dynein heavy chain (cDhc64C) and the p150Glued (Glued) component of the dynactin complex with the use of genetic techniques in Drosophila. cDhc64C and Glued mutations disrupt fast organelle transport in both directions. The mutant phenotypes, larval posterior paralysis and axonal swellings filled with retrograde and anterograde cargoes, were similar to those caused by kinesin mutations. Why do specific disruptions of unidirectional motor systems cause bidirectional defects? Direct protein interactions of kinesin with dynein heavy chain and p150Glued were not detected. However, strong dominant genetic interactions between kinesin, dynein, and dynactin complex mutations in axonal transport were observed. The genetic interactions between kinesin and either Glued or cDhc64C mutations were stronger than those between Glued and cDhc64C mutations themselves. The shared bidirectional disruption phenotypes and the dominant genetic interactions demonstrate that cytoplasmic dynein, the dynactin complex, and conventional kinesin are interdependent in fast axonal transport.

Herpesviruses use bidirectional fast-axonal transport to spread in sensory neurons

Alpha herpesviruses infect the vertebrate nervous system resulting in either mild recurrent lesions in mucosal epithelia or fatal encephalitis. Movement of virions within the nervous system is a critical factor in the outcome of infection; however, the dynamics of individual virion transport have never been assessed. Here we visualized and tracked individual viral capsids as they moved in axons away from infected neuronal cell bodies in culture. The observed movement was compatible with fast axonal flow mediated by multiple microtubule motors. Capsids accumulated at axon terminals, suggesting that spread from infected neurons required cell contact.

Cytoplasmic dynein is associated with slow axonal transport.

Neuronal function is dependent on the transport of materials from the cell body to the synapse via anterograde axonal transport. Anterograde axonal transport consists of several components that differ in both rate and protein composition. In fast transport, membranous organelles are moved along microtubules by the motor protein kinesin. The cytoskeleton and the cytomatrix proteins move in the two components of slow transport. While the mechanisms underlying slow transport are unknown, it has been hypothesized that the movement of microtubules in slow transport is generated by sliding. To determine whether dynein, a motor protein that causes microtubule sliding in flagella, may play a role in slow axonal transport, we identified the transport rate components with which cytoplasmic dynein is associated in rat optic nerve. Nearly 80% of the anterogradely moving dynein was associated with slow transport, whereas only approximately 15% of the dynein was associated with the membranous organelles of anterograde fast axonal transport. A segmental analysis of the transport of dynein through contiguous regions of the optic nerve and tract showed that dynein is associated with the microfilaments and other proteins of slow component b. Dynein from this transport component has the capacity to bind microtubules in vitro. These results are consistent with the hypothesis that cytoplasmic dynein generates the movement of microtubules in slow axonal transport. A model is presented to illustrate how dynein attached to the slow component b complex of proteins is appropriately positioned to generate force of the correct polarity to slide microtubules down the axon.

Retrograde axonal transport of glial cell line-derived neurotrophic factor in the adult nigrostriatal system suggests a trophic role in the adult.

The recently cloned, distant member of the transforming growth factor beta (TGF-beta) family, glial cell line-derived neurotrophic factor (GDNF), has potent trophic actions on fetal mesencephalic dopamine neurons. GDNF also has protective and restorative activity on adult mesencephalic dopaminergic neurons and potently protects motoneurons from axotomy-induced cell death. However, evidence for a role for endogenous GDNF as a target-derived trophic factor in adult midbrain dopaminergic circuits requires documentation of specific transport from the sites of synthesis in the target areas to the nerve cell bodies themselves. Here, we demonstrate that GDNF is retrogradely transported by mesencephalic dopamine neurons of the nigrostriatal pathway. The pattern of retrograde transport following intrastriatal injections indicates that there may be subpopulations of neurons that are GDNF responsive. Retrograde axonal transport of biologically active 125I-labeled GDNF was inhibited by an excess of unlabeled GDNF but not by an excess of cytochrome c. Specificity was further documented by demonstrating that another TGF-beta family member, TGF-beta 1, did not appear to affect retrograde transport. Retrograde transport was also demonstrated by immunohistochemistry by using intrastriatal injections of unlabeled GDNF. GDNF immunoreactivity was found specifically in dopamine nerve cell bodies of the substantia nigra pars compacta distributed in granules in the soma and proximal dendrites. Our data implicate a specific receptor-mediated uptake mechanism operating in the adult. Taken together, the present findings suggest that GDNF acts endogenously as a target-derived physiological survival/maintenance factor for dopaminergic neurons.

Transport of cytoskeletal elements in the squid giant axon.

In order to explore how cytoskeletal proteins are moved by axonal transport, we injected fluorescent microtubules and actin filaments as well as exogenous particulates into squid giant axons and observed their movements by confocal microscopy. The squid giant axon is large enough to allow even cytoskeletal assemblies to be injected without damaging the axon or its transport mechanisms. Negatively charged, 10- to 500-nm beads and large dextrans moved down the axon, whereas small (70 kDa) dextrans diffused in all directions and 1000-nm beads did not move. Only particles with negative charge were transported. Microtubules and actin filaments, which have net negative charges, made saltatory movements down the axon, resulting in a net rate approximating that previously shown for slow transport of cytoskeletal elements. The present observations suggest that particle size and charge determine which materials are transported down the axon.

Pathogens penetrating the central nervous system: Infection pathways and the cellular and molecular mechanisms of invasion

The brain is well protected against microbial invasion by cellular barriers, such as the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB). In addition, cells within the central nervous system (CNS) are capable of producing an immune response against invading pathogens. Nonetheless, a range of pathogenic microbes make their way to the CNS, and the resulting infections can cause significant morbidity and mortality. Bacteria, amoebae, fungi, and viruses are capable of CNS invasion, with the latter using axonal transport as a common route of infection. In this review, we compare the mechanisms by which bacterial pathogens reach the CNS and infect the brain. In particular, we focus on recent data regarding mechanisms of bacterial translocation from the nasal mucosa to the brain, which represents a little explored pathway of bacterial invasion but has been proposed as being particularly important in explaining how infection with Burkholderia pseudomallei can result in melioidosis encephalomyelitis.