Arrival, reversal, and departure of neurofilaments at the tips of growing axons

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by 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.     

Rapid intermittent movement of axonal neurofilaments observed by fluorescence photobleaching

Observations on naturally occurring gaps in the axonal neurofilament array of cultured neurons have demonstrated that neurofilament polymers move along axons in a rapid, intermittent, and highly asynchronous manner. In contrast, studies on axonal neurofilaments using laser photobleaching have not detected movement. Here, we describe a modified photobleaching strategy that does permit the direct observation of neurofilament movement. Axons of cultured neurons expressing GFP-tagged neurofilament protein were bleached by excitation with the mercury arc lamp of a conventional epifluorescence microscope for 12-60 s. The length of the bleached region ranged from 10 to 60 microm. By bleaching thin axons, which have relatively few neurofilaments, we were able to reduce the fluorescent intensity enough to allow the detection of neurofilaments that moved in from the surrounding unbleached regions. Time-lapse imaging at short intervals revealed rapid, intermittent, and highly asynchronous movement of fluorescent filaments through the bleached regions at peak rates of up to 2.8 microm/s. The kinetics of movement were very similar to our previous observations on neurofilaments moving through naturally occurring gaps, which indicates that the movement was not impaired by the photobleaching process. These results demonstrate that fluorescence photobleaching can be used to study the slow axonal transport of cytoskeletal polymers, but only if the experimental strategy is designed to ensure that rapid asynchronous movements can be detected. This may explain the failure of previous photobleaching studies to reveal the movement of neurofilament proteins and other cytoskeletal proteins in axons.     

Differential spatial and temporal expression of two type III intermediate filament proteins in olfactory receptor neurons

Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons.     

Control of axonal caliber by neurofilament transport

The role of neurofilaments, the intermediate filaments of nerve cells, has been conjectural. Previous morphological studies have suggested a close relationship between neurofilament content and axonal caliber. In this study, the regenerating neuron was used as a model system for testing the hypotheses that neurofilaments are intrinsic determinants of axonal caliber, and that neurofilament content is controlled by the axonal transport of neurofilaments. This system was chosen because previous studies had shown that, after axotomy, axonal caliber was reduced within the proximal stump of the regenerating nerve and, because the relative amount of neurofilament protein undergoing axonal transport in regenerating axons was selectively reduced. The relationship between axonal caliber and neurofilament number was examined in a systematic fashion in both regenerating and control motor axons in rat L5 ventral root. Reconstruction of the spatial and temporal sequences of axonal atrophy in the proximal stump after axotomy showed that reductions in axonal caliber were first detected in the most proximal region of the root and subsequently progressed in a proximal-to-distal direction at a rate of 1.7 mm/day, which is identical to the rate of neurofilament transport in these neurons. Quantitative ultrastructural studies showed that these reductions in caliber correlated with a proportional decrease in the number of axonal neurofilaments but not microtubules. These results support the hypotheses that neurofilament content is a major intrinsic determinant of axonal caliber and that neurofilament content is controlled by the axonal transport of neurofilaments. On this basis, we suggest a role for neurofilaments in the control of axonal volume.     

Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.     

Identification and Cytoprotective Function of a Novel Nestin Isoform,Nes-S,in Dorsal Root Ganglia Neurons

In this study, the first nestin isoform, Nes-S, was identified in neurons of dorsal root ganglia (DRG) of adult rats. Nes-S cannot form filaments by itself in cytoplasmic intermediate filament-free SW13 cells. Instead, it co-assembles into filaments with vimentin when transfected into vimentin⁺ SW13 cells, and with peripherin and neurofilament proteins when transfected into N2a cells. In primary DRG neurons, endogenous Nes-S co-assembles with peripherin and neurofilament proteins. The expression of Nes-S first appears in DRG at postnatal day 5 and persists to adulthood. Among the adult tissues we examined, the expression of Nes-S is restricted to the sensory and motor neurons. Finally, exogenous Nes-S enhances viability when transfected into N2a cells, and knockdown of endogenous Nes-S impairs the survival of DRG neurons in primary cultures. Taken together, Nes-S is a new neuronal intermediate filament protein that exerts a cytoprotective function in mature sensory and motor neurons.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Contactin 1 knockdown in the hindbrain induces abnormal development of the trigeminal sensory nerve in <Emphasis Type="Italic">Xenopus</Emphasis> embryos

In Xenopus tailbud embryos, the mandibular branch of trigeminal sensory nerve has a transient pathway innervating the cement gland. This pathway is settled by pioneer neurons in the trigeminal ganglion and along which extend later-growing axons from the trigeminal ganglion and the hindbrain. Axons in this branch express a neuronal recognition molecule, Contactin 1, from the initial stage of its outgrowth in early tailbud embryos and form a tightly joined, strongly Contactin 1-positive fascicle in the later stages. When the expression vector encoding the enhanced green fluorescent protein was electrotransfected into the brain neurons of early tailbud embryos, the fluorescence was detected in the hindbrain and the trigeminal nerve at late tailbud stages. Cotransfection of antisense vector caused knockdown of Contactin 1 concurrent with defasciculation and misguidance of the sensory axons in the trigeminal mandibular branch. The results suggest that Contactin 1 is required for the growing axon of hindbrain sensory neurons to recognize and follow the pathway settled by the pioneer neurons.     

O-GlcNAc modification of radial glial vimentin filaments in the developing chick brain

We examined the post-translational modification of intracellular proteins by β-O-linked N-acetylglucosamine (O-GlcNAc) with regard to neurofilament phosphorylation in the developing chick optic tectum. A regulated developmental pattern of O-GlcNAcylation was discovered in the developing brain. Most notably, discernible staining occurs along radial glial filaments but not along neuronal filaments in vivo. Immunohistochemical analyses in sections of progressive stages of development suggest upregulation of O-GlcNAc in the ependyma, tectofugal neuron bodies, and radial glial processes, but not in axons. In contrast, double-label immunostaining of monolayer cultures made from dissociated embryonic day (E) 7 optic tecta revealed O-GlcNAcylation of most axons. Labeling of brain sections together with Western blot analyses showed O-GlcNAc modification of a few discrete proteins throughout development, and suggested vimentin as the protein in radial glia. Immunoprecipitation of vimentin from E9 whole brain lysates confirmed O-GlcNAcylation of vimentin in development. These results indicate a regulated pattern of O-GlcNAc modification of vimentin filaments, which in turn suggests a role for O-GlcNAc-modified intermediate filaments in radial glia, but not in neurons during brain development. The control mechanisms that regulate this pattern in vivo, however, are disrupted when cells are placed in vitro.     

Neurofilament functions in health and disease.

Transgenic approaches have recently been used to investigate the functions of neuronal intermediate filaments. Gene knockout studies have demonstrated that neurofilaments are not required for axogenesis and that individual neurofilament proteins play distinct roles in filament assembly and in the radial growth of axons. The involvement of neurofilaments in disease is supported by the discovery of novel mutations in the neurofilament heavy gene from cases of amyotrophic lateral sclerosis and by reports of neuronal death in mouse models expressing neurofilament and alpha-internexin transgenes. However, mouse studies have shown that axonal neurofilaments are not required for pathogenesis caused by mutations in superoxide dismutase and that increasing perikaryal levels of neurofilament proteins may even confer protection in this disease.     

Functions of intermediate filaments in neuronal development and disease

Five major types of intermediate filament (IF) proteins are expressed in mature neurons: the three neurofilament proteins (NF-L, NF-M, and NF-H), alpha-internexin, and peripherin. While the differential expression of IF genes during embryonic development suggests potential functions of these proteins in axogenesis, none of the IF gene knockout experiments in mice caused gross developmental defects of the nervous system. Yet, deficiencies in neuronal IF proteins are not completely innocuous. Substantial developmental loss of motor axons was detected in mice lacking NF-L and in double knockout NF-M;NF-H mice, supporting the view of a role for IFs in axon stabilization. Moreover, the absence of peripherin resulted in approximately 30% loss of small sensory axons. Mice lacking NF-L had a scarcity of IF structures and exhibited a severe axonal hypotrophy, causing up to 50% reduction in conduction velocity, a feature that would be very detrimental for large animal species. Unexpectedly, the NF-M rather than NF-H protein turned out to be required for proper radial growth of large myelinated axons. Studies with transgenic mice suggest that some types of IF accumulations, reminiscent of those found in amyotrophic lateral sclerosis (ALS), can have deleterious effects and even cause neurodegeneration. Additional evidence for the involvement of IFs in pathogenesis came from the recent discovery of neurofilament gene mutations linked to ALS and Charcot-Marie-Tooth disease (CMT2E). Conversely, we discuss how certain types of perikaryal neurofilament aggregates might confer protection in motor neuron disease.     

An intrinsic neuronal defect operates in dystonia musculorum: a study of dt/dt<==>+/+ chimeras.

In the mouse mutant dystonia musculorum (dt), peripheral and central sensory axons develop focal swellings and degenerate. To identify the primary cellular target of the mutation, we have analyzed the spinal cords of dt/dt<==>+/+ aggregation chimeras. In these chimeras, characteristic swellings appeared only on the axons of mutant genotype neurons; the axons of wild-type neurons, identified by their expression of a transgene-encoded human neurofilament protein, were normal. This direct correlation of genotype and phenotype indicates that the dt mutation acts via a mechanism intrinsic to affected neurons. In addition, we show here that the dt mutation leads to a disorder of neurofilament processing in which phosphorylated neurofilament epitopes accumulate inappropriately in neuronal perikarya.     

Involvement of CTP:phosphocholine cytidylyltransferase-β2 in axonal phosphatidylcholine synthesis and branching of neurons

In the brain, phosphatidylcholine (PC) is synthesized by the CDP-choline pathway in which the rate-limiting step is catalyzed by two isoforms of CTP:phosphocholine cytidylyltransferase (CT): CTα and CTβ2. In mice, CTβ2 mRNA is more highly expressed in the brain than in other tissues, and several observations suggest that CTβ2 plays an important role in the nervous system. We, therefore, investigated the importance of CTβ2 for PC synthesis as well as for axon formation, growth and branching of primary sympathetic neurons. We show that in cultured primary neurons nerve growth factor increases the amount of CTβ2, but not CTα, mRNA and protein. The brains of mice lacking CTβ2 had normal PC content despite having 35% lower CT activity than wild-type brains. CTβ2 mRNA and protein are abundant in distal axons of mouse sympathetic neurons whereas CTα mRNA and protein were not detected. Moreover, CTβ2 deficiency in distal axons reduced the incorporation of [(3)H]choline into PC by 95% whereas PC synthesis in cell bodies/proximal axons was unaltered. These data suggest that CTβ2 is the major CT isoform involved in PC synthesis in axons. Axons of CTβ2-deficient sympathetic neurons contained 32% fewer branch points than did wild-type neurons although the number of axons/neuron and the rate of axon extension were the same as in wild-type neurons. We conclude that in distal axons of primary sympathetic neurons CTβ2 is a major contributor to PC synthesis and promotes axon branching, whereas CTα appears to be the major CT isoform involved in PC synthesis in cell bodies/proximal axons.     

Singar1, a novel RUN domain-containing protein, suppresses formation of surplus axons for neuronal polarity

Although neuronal functions depend on their robust polarity, the mechanisms that ensure generation and maintenance of only a single axon remain poorly understood. Using highly sensitive two-dimensional electrophoresis-based proteomics, we identified here a novel protein, single axon-related (singar)1/KIAA0871/RPIPx/RUFY3, which contains a RUN domain and is predominantly expressed in the brain. Singar1 expression became up-regulated during polarization of cultured hippocampal neurons and remained at high levels thereafter. Singar1 was diffusely localized in hippocampal neurons and moderately accumulated in growth cones of minor processes and axons. Overexpression of singar1 did not affect normal neuronal polarization but suppressed the formation of surplus axons induced by excess levels of shootin1, a recently identified protein located upstream of phosphoinositide-3-kinase and involved in neuronal polarization. Conversely, reduction of the expression of singar1 and its splicing variant singar2 by RNA interference led to an increase in the population of neurons bearing surplus axons, in a phosphoinositide-3-kinase-dependent manner. Overexpression of singar2 did not suppress the formation of surplus axons induced by shootin1. We propose that singar1 ensures the robustness of neuronal polarity by suppressing formation of surplus axons.     

Posttranslational modification of neurofilament polypeptides in rabbit retina

Three polypeptides that compose neurofilaments, designated H, M, and L, are synthesized in the cell bodies of neurons and subsequently conveyed down their axons by the process of slow axonal transport. The axonal form of H, which is a component of the cross bridges between the neurofilaments, is antigenically different from the form in the cell bodies and dendrites. To understand how this special form of H is directed to the axon, and more generally how intracellular differentiation is established and maintained by the selective delivery of different molecular species to different compartments of a cell, we have studied the events that occur immediately after the synthesis of the three neurofilament polypeptides in the retinas of rabbits. We observed that H and M are synthesized in the retina as precursor polypeptides, EH and EM, that migrate markedly faster on SDS polyacrylamide gels than their mature axonal forms. The maturation of these precursors requires more than one day and appears to involve their phosphorylation. Only the electrophoretically mature forms appear in the axons of the retinal ganglion cells in the optic nerve. We consider the following interpretation of these observations. Shortly after they are translated in the cell body, the neurofilament polypeptides become phosphorylated at multiple sites. However, only after they have moved a distance of several hundred micrometers down the axon, H and M are phosphorylated at additional sites, causing their conformation or binding properties to change. This change, which is reflected in the reduction of their electrophoretic mobility and the appearance of new antigenic determinants, may function to alter the H-mediated crossbridges and produces the morphological and structural properties of the neurofilament lattice that is characteristic of axons.     

The myosin Va head domain binds to the neurofilament-L rod and modulates endoplasmic reticulum (ER) content and distribution within axons

The neurofilament light subunit (NF-L) binds to myosin Va (Myo Va) in neurons but the sites of interaction and functional significance are not clear. We show by deletion analysis that motor domain of Myo Va binds to the NF-L rod domain that forms the NF backbone. Loss of NF-L and Myo Va binding from axons significantly reduces the axonal content of ER, and redistributes ER to the periphery of axon. Our data are consistent with a novel function for NFs as a scaffold in axons for maintaining the content and proper distribution of vesicular organelles, mediated in part by Myo Va. Based on observations that the Myo Va motor domain binds to intermediate filament (IF) proteins of several classes, Myo Va interactions with IFs may serve similar roles in organizing organelle topography in different cell types.     

Dcc regulates asymmetric outgrowth of forebrain neurons in zebrafish

The guidance receptor DCC (deleted in colorectal cancer) ortholog UNC-40 regulates neuronal asymmetry development in Caenorhabditis elegans, but it is not known whether DCC plays a role in the specification of neuronal polarity in vertebrates. To examine the roles of DCC in neuronal asymmetry regulation in vertebrates, we studied zebrafish anterior dorsal telencephalon (ADt) neuronal axons. We generated transgenic zebrafish animals expressing the photo-convertible fluorescent protein Kaede in ADt neurons and then photo-converted Kaede to label specifically the ADt neuron axons. We found that ADt axons normally project ventrally. Knock down of Dcc function by injecting antisense morpholino oligonucleotides caused the ADt neurons to project axons dorsally. To examine the axon projection pattern of individual ADt neurons, we labeled single ADt neurons using a forebrain-specific promoter to drive fluorescent protein expression. We found that individual ADt neurons projected axons dorsally or formed multiple processes after morpholino knock down of Dcc function. We further found that knock down of the Dcc ligand, Netrin1, also caused ADt neurons to project axons dorsally. Knockdown of Neogenin1, a guidance receptor closely related to Dcc, enhanced the formation of aberrant dorsal axons in embryos injected with Dcc morpholino. These experiments provide the first evidence that Dcc regulates polarized axon initiation and asymmetric outgrowth of forebrain neurons in vertebrates.     

Role of Electrical Activity in Horizontal Axon Growth in the Developing Cortex: A Time-Lapse Study Using Optogenetic Stimulation

During development, layer 2/3 neurons in the neocortex extend their axons horizontally, within the same layers, and stop growing at appropriate locations to form branches and synaptic connections. Firing and synaptic activity are thought to be involved in this process, but how neuronal activity regulates axonal growth is not clear. Here, we studied axonal growth of layer 2/3 neurons by exciting cell bodies or axonal processes in organotypic slice cultures of the rat cortex. For neuronal stimulation and morphological observation, plasmids encoding channelrhodopsin-2 (ChR2) and DsRed were coelectroporated into a small number of layer 2/3 cells. Firing activity induced by photostimulation (475 nm) was confirmed by whole-cell patch recording. Axonal growth was observed by time-lapse confocal microscopy, using a different excitation wavelength (560 nm), at 10–20-min intervals for several hours. During the first week in vitro, when spontaneous neuronal activity is low, DsRed- and ChR2-expressing axons grew at a constant rate. When high-frequency photostimulation (4 or 10 Hz) for 1 min was applied to the soma or axon, most axons paused in their growth. In contrast, lower-frequency stimulation did not elicit this pause behavior. Moreover, in the presence of tetrodotoxin, even high-frequency stimulation did not cause axonal growth to pause. These results indicate that increasing firing activity during development suppresses axon growth, suggesting the importance of neuronal activity for the formation of horizontal connections.     

Temporal and spatial modulation of a cytoskeletal antigen during peripheral axonal pathfinding.

A neuron-specific cytoskeletal antigen (5E10), whose expression pattern during initial motoneuron outgrowth into the chick limb suggests that it is playing a role in axon guidance, is described. This antigen, which was shown to be a phosphorylated epitope, probably of the intermediate weight neurofilament protein (NF-M), exhibits a highly stereotyped and spatially heterogeneous pattern of expression. The point of onset of expression, which was abrupt and occurred in the distal axon and base of the growth cone, differed between groups of neurons that projected to different targets. Specifically, expression occurred from positions where previous perturbation experiments suggested that the axons in question would begin responding to specific guidance cues, and it remained high along the axon from this point to the target. Expression of this antigen could also be induced in cultured motoneurons by activating several second messenger systems.