Neuronal Polarization: Old Cells Can Learn New Tricks

The Journal of Neuroscience, 1989

Biological Reviews, 1979

Journal of Neurocytology, 1992

If one end of a segment of peripheral nerve is inserted into the brain or spinal cord, neuronal perikarya in the vicinity of the graft tip can be labelled with retrogradely transported tracers applied to the distal end of the graft several weeks later, showing that CNS axons can regenerate into and along such grafts. We have used transmission EM to examine some of the cellular responses that underlie this regenerative phenomenon, particularly its early stages. Segments of autologous peroneal or tibial nerve were inserted vertically into the thalamus of anaesthetized adult albino rats. The distal end of the graft was left beneath the scalp. Between five days and two months later the animals were killed and the brains prepared for ultrastructural study. Semi-thin and thin sections through the graft and surrounding brain were examined at two levels 6-7 mm apart in all animals: close to the tip of the graft in the thalamus (proximal graft) and at the top of the cerebral cortex (distal graft). In another series of animals with similar grafts, horseradish peroxidase was applied to the distal end of the graft 2448 h before death. Examination by LM of appropriately processed serial coronal sections of the brains from these animals confirmed that up to several hundred neurons were retrogradely labelled in the thalamus, particularly in the thalamic reticular nucleus.

Trends in Neurosciences, 2000

Neural geometry is the major factor that determines connectivity and, possibly, functional output from a nervous system. Recently some of the proteins and pathways involved in specific modes of branch formation or maintenance, or both, have been described.To a variable extent, dendrites and axon collaterals can be viewed as dynamic structures subject to fine modulation that can result either in further growth or retraction. Each form of branching results from specific molecular mechanisms. Cell-internal, substrate-derived factors and functional activity, however, can often differ in their effect according to cell type and physiological context at the site of branch formation. Neural branching is not a linear process but an integrative one that takes place in a microenvironment where we have only a limited experimental access. To attain a coherent mechanism for this phenomenon, quantitative in situ data on the proteins involved and their interactions will be required.

Neuroscience, 1994

Microtubule associated proteins play a central role in the control of axon growth. We have used immunohistochemical techniques to establish which microtubule-associated proteins are present in the rat hindlimb spinal cord, dorsal root ganglia and peripheral nerves during axonal growth during embryogenesis, in adulthood, and during regeneration of crushed sciatic nerves. During embryogenesis microtubule-associated protein-lb and tau are present in all neurons and axons, microtubule-associated protein-2 is present in neurons but not in axons, and there is no microtubule-associated protein-la. In adults, microtubule-associated protein-la and microtubule-associated protein-lb are present in all sciatic nerve axons and in motor and dorsal root ganglion neurons, Tau, in its adult form, is present in many fine probably sensory axons, but not in most larger axons, and in motor and sensory neurons. Microtubule-associated protein-2 is present only in neurons. During regeneration the pattern of microtubule-associated protein expression retains the adult pattern. All regenerating axons contain microtubule-associated protein-la and microtubule-associated protein-lb, none contain microtubuleassociated protein-2, and a subset of fine axons contain tau. There is no detectable change in microtubule-associated protein expression by motoneurons. While axons are clearly able to regenerate without either microtubule-associated protein-2 or tau, tau containing axons appear to regenerate faster than those which lack it. It is possible that the failure of neurons to recapitulate the embryonic pattern of microtubule-assoaated protein expression during regeneratron could be a reason why regenerative axon growth is slower and less vigorous than axon growth in embryos

Journal of Neurotrauma, 1998

In animal models of human diffuse axonal injury, axonal swellings leading to secondary axotomy occur between 2 and 6 h after injury. But, analysis of cytoskeletal changes associated with secondary axotomy has not been undertaken. We have carried out a quantitative analysis of cytoskeletal changes in a model of diffuse axonal injury 4 h after stretch-injury to adult guinea-pig optic nerves. The major site of axonal damage was the middle portion of the nerve. There was a statistically significant increase in the proportion of small axons with a diameter of 0.5 m and smaller in which there was compaction of neurofilaments. Axons with a diameter greater than 2.0 m demonstrated an increased spacing between cytoskeletal elements throughout the length of the nerve. However, in the middle segment of the nerve these larger axons demonstrated two different types of response. Either, where periaxonal spaces occurred, there was a reduction in axonal calibre, compaction of neurofilaments but no change in their number, and a loss of microtubules. Or, where intramyelinic spaces occurred there was an increased spacing between neurofilaments and microtubules with a significant loss in the number of both. Longitudinal sections showed foci of compaction of neurofilaments interspersed between regions where axonal structure was apparently normal. Neurofilament compaction was correlated with disruption of the axolemma at these foci present some hours after injury. We suggest that the time course of these axonal cytoskeletal changes after stretch-injury to central axons is shorter than those changes documented to occur during Wallerian degeneration.

BioArchitecture, 2014

The Journal of Neuroscience, 2007

Axons in the CNS do not regrow after injury, whereas lesioned axons in the peripheral nervous system (PNS) regenerate. Lesioned CNS axons form characteristic swellings at their tips known as retraction bulbs, which are the nongrowing counterparts of growth cones. Although much progress has been made in identifying intracellular and molecular mechanisms that regulate growth cone locomotion and axonal elongation, a comprehensive understanding of how retraction bulbs form and why they are unable to grow is still elusive. Here we report the analysis of the morphological and intracellular responses of injured axons in the CNS compared with those in the PNS. We show that retraction bulbs of injured CNS axons increase in size over time, whereas growth cones of injured PNS axons remain constant. Retraction bulbs contain a disorganized microtubule network, whereas growth cones possess the typical bundling of microtubules. Using in vivo imaging, we find that pharmacological disruption of microtubules in growth cones transforms them into retraction bulb-like structures whose growth is inhibited. Correspondingly, microtubule destabilization of sensory neurons in cell culture induces retraction bulb formation. Conversely, microtubule stabilization prevents the formation of retraction bulbs and decreases axonal degeneration in vivo. Finally, microtubule stabilization enhances the growth capacity of CNS neurons cultured on myelin. Thus, the stability and organization of microtubules define the fate of lesioned axonal stumps to become either advancing growth cones or nongrowing retraction bulbs. Our data pinpoint microtubules as a key regulatory target for axonal regeneration.

Molecular Biology of the Cell, 2004

We have investigated the movement of GFP-tagged neurofilaments at the distal ends of growing axons using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that (1) growth cones are a preferential site of neurofilament reversal in distal axons, (2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, (3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and (4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency. dissociated from the superior cervical ganglion of neonatal rats typically grow at rates of 4-30 µm/hour (0.001-0.008 µm/s; , depending on the substrate, yet neurofilaments in these neurons move at average rates (excluding pauses) of 0.4-0.7 µm/s . This suggests that anterogradely moving neurofilaments in the distal regions of growing axons may catch up with the 5 advancing growth cone. Previous studies did not address this hypothesis because they were all performed in intermediate regions of the axon, far from the growth cone. Thus, in the present study, we investigated neurofilament transport at the tips of growing axons. We found that most anterograde neurofilaments in the distal axon catch up with and enter the growth cone. In addition, and to our surprise, some of these filaments subsequently reverse direction, exit the growth cone, and move back towards the cell body. Thus local mechanisms at the axon tip can alter the direction of neurofilament movement, presumably by altering the activity of the neurofilament motors or their affinity for their cargo.

The identities of axons and dendrites are acquired through the self-organization of distinct microtubule (MT) orientations during neuronal polarization. The axon is generally characterized by a uniform MT orientation with all plus-ends pointing outward to the neurite terminal ('plus-end-out' pattern). On the other hand, the MT orientation pattern in the dendrites depends on species: vertebrate dendrites have a mixed alignment with both plus and minus ends facing either the terminal or the cell body ('mixed' pattern), whereas invertebrate dendrites have a 'minus-end-out' pattern. However, how MT organizations are developed in the axon and the dendrites is largely unknown. To investigate the mechanism of MT organization, we developed a biophysical model of MT kinetics, consisting of polymerization/depolymerization and MT catastrophe coupled with neurite outgrowth. The model simulation showed that the MT orientation can be controlled mainly by the speed of neurite growth and the hydrolysis rate. With a low hydrolysis rate, vertebrate plus-end-out and mixed microtubule patterns emerged in fast-and slow-growing neurites, respectively. In contrast, with a high hydrolysis rate, invertebrate plus-end-out and minus-end-out microtubule patterns emerged in fast-and slowgrowing neurites, respectively. Thus, our model can provide a unified understanding of distinct microtubule organizations by simply changing the parameters.

2012

One of the striking features of the injured central nervous system (CNS) is the failure of severed axons to adequately regenerate to restore loss of function. This was initially believed to be caused by an intrinsic inability of injured axons to sprout regenerative processes. However, the seminal studies of Albert Aguayo and others using peripheral or cellular tissue grafts transplanted into the lesioned spinal cord have clearly demonstrated that the environment of the injured CNS is a critical determinant of whether injured axons can regenerate . The molecular determinates of the inhibitory CNS environment are now well-understood, with major players being myelin-associated molecules (such as nogo, myelin-associated glycoprotein) and chondroitin sulphate proteoglycans .

Proceedings of the …, 2001

Experimental evidence suggests that microfilaments and microtubules play contrasting roles in regulating the balance between motility and stability in neuronal structures. Actin-containing microfilaments are associated with structural plasticity, both during development when their dynamic activity drives the exploratory activity of growth cones and after circuit formation when the actin-rich dendritic spines of excitatory synapses retain a capacity for rapid changes in morphology. By contrast, microtubules predominate in axonal and dendritic processes, which appear to be morphologically relatively more stable. To compare the cytoplasmic distributions and dynamics of microfilaments and microtubules we made time-lapse recordings of actin or the microtubuleassociated protein 2 tagged with green fluorescent protein in neurons growing in dispersed culture or in tissue slices from transgenic mice. The results complement existing evidence indicating that the high concentrations of actin present in dendritic spines is a specialization for morphological plasticity. By contrast, microtubule-associated protein 2 is limited to the shafts of dendrites where time-lapse recordings show little evidence for dynamic activity. A parallel exists between the partitioning of microfilaments and microtubules in motile and stable domains of growing processes during development and between dendrite shafts and spines at excitatory synapses in established neuronal circuits. These data thus suggest a mechanism, conserved through development and adulthood, in which the differential dynamics of actin and microtubules determine the plasticity of neuronal structures.

Cellular Migration and Formation of Neuronal Connections, 2013

The Journal of Neuroscience

The Journal of cell biology, 1992

Abstract. We have investigated the sites of microtu-bule (MT) assembly in neurons during axon growth by taking advantage of the relationship between the pro-portion of tyrosinated c~-tubulin (tyr-tubulin) in MTs and their age. Specifically, young (newly assembled) MTs contain ...