Neurofilament phosphorylation in peripheral nerve regeneration

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.     

Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: in vivo and in vitro study with monoclonal antibodies

Neurofilament phosphorylation in rat nervous system development was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes in tissue sections and in primary dissociated cultures. The antibodies either decorated neurofilaments shortly after their appearance or after a considerable delay (from 4 to 9 days in vivo and from 12 to 27 days in vitro), thus suggesting the existence of at least two classes of phosphorylated epitopes. With most antibodies there was a good correlation between in vivo and in vitro findings as to the early or late appearance of phosphorylated epitopes. Monoclonal NE14 was the main exception in that immunoreactivity with this antibody was present in 1-day cultures, while it only occurred 4 days after the first appearance of neurofilaments in vivo. The effect of phosphorylation on neurofilament structure and function remains to be determined. Neurofilament expression is an early phenomenon in ontogeny coinciding with neuronal differentiation. It is possible that late phosphorylation events may stabilize the axonal cytoskeleton following the massive loss of axons that occurs in several fiber tracts during late fetal and neonatal life.     

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.     

Altered tubulin and neurofilament expression and impaired axonal growth in diabetic nerve regeneration

Cytoskeletal protein expression in sensory neurons and sciatic nerve axonal growth were examined in type 1 diabetic BB/Wor rats after sciatic nerve crush injury. Diabetic male rats were subjected to sciatic nerve crush at 6 wk of diabetes. L4 and L5 dorsal root ganglia (DRG) mRNA expression of low and medium molecular weight neurofilaments (NF-L, NF-M), betaII- and betaIII-tubulin as well as protein expression of NF-L, NF-M, and beta-tubulin were examined at various time points following crush injury and compared with age- and sex-matched non-diabetic BB/Wor rats. Steady state mRNA expression of NF-L, NF-M, betaII- and betaIII-tubulin were decreased in diabetic DRG. NF-L and NF-M proteins were also decreased in DRG of uncrushed diabetic animals. After crush injury, betaII- and betaIII-tubulin mRNA were upregulated in control animals at day 2 and day 6, respectively, and beta-tubulin protein showed similarly increased expression after crush injury, while such upregulations did not occur in diabetic animals. Conversely, mRNA and protein expressions of NF-L, NF-M were downregulated to a lesser extent in diabetic animals compared to control rats. These changes were associated with impaired axonal elongation and caliber growth of regenerating fibers in diabetic rats. We propose that upregulation of tubulin has a negative feedback on NF expression in response to nerve injury, as seen in control rats. The absence of this upregulation in diabetic animals may impair its regulatory effect on NF expression and contribute to perturbed nerve regeneration seen in diabetic nerve.     

Early history of mammalian nerve regeneration

The history of mammalian nerve regeneration research was reviewed up until the time of Ramón y Cajal. Progress in both peripheral and CNS regeneration was discussed in relation to advances in neurobiology and histology and to the auto-regeneration controversy which persisted throughout the latter half of the nineteenth century. The positions of the monogenists and polygenists were presented and the contributions which sprang from attempts to resolve the controversy were explored. The early investigators developed basic concepts and defined the problems of regeneration research. Their efforts contributed significantly to our understanding of the behavior of neurons following axotomy and laid the foundation for modern investigators in morphological plasticity of neurons.     

Ultrastructural appearance of intentionally frustrated axonal regeneration in rat sciatic nerve

The ultrastructural appearance of axons regenerating after crush injury was examined in rat sciatic nerves in which proximodistal growth was interrupted (frustrated regeneration) by placement of a tight ligature 1 cm distal to the original crush injury, and in nerves lacking a distal tie (unimpeded regeneration). Examination of unimpeded regenerating axons four and seven days after injury showed minute axonal sprouts as well as scattered dilated (2-10 microns) axonal profiles containing large numbers of anastomosing tubulovesicular elements and vacuoles. These dilated profiles were consistent with the appearance of growth cones, the motile tips of regenerating axons, as described in various in vivo and in vitro systems. The ultrastructural appearance of regenerating axons accumulating proximal to a frustrating tie was a function of time after arrival at the ligature. At the earliest time examined (one week) large numbers of axonal profiles accumulated at the ligature which were qualitatively similar to growth cones seen in unimpeded regeneration, although slightly larger in diameter. With time, the uniform population of growth cones proximal to the frustrating ligature was replaced by dilated axons containing large numbers of neurofilaments, dense collections of heterogeneous membranous organelles, or delicate to coarse tubulovesicular elements admixed with a variety of subcellular organelles. Rare examples of axonal dystrophy were demonstrated after chronic frustration of regeneration; however, they represented only a small percentage (less than 1%) of frustrated axons. Frustrated regeneration was accompanied by scattered examples of demyelination/remyelination of large axons proximal to the ligature as well as transperineurial growth of axons as microfascicles to escape the site of ligation.     

Regulation of neurofilament dynamics by phosphorylation

Neurofilament (NF) phosphorylation has long been considered to regulate axonal transport rate and in doing so to provide stability to mature axons. Studies utilizing mice in which the C-terminal region of NF subunits (which contains the vast majority of phosphorylation sites) has been deleted has prompted an ongoing challenge to this hypothesis. We evaluate the collective evidence to date for and against a role for NF C-terminal phosphorylation in regulation of axonal transport and in providing structural support for axons, including some novel studies from our laboratory. We present a few suggestions for further experimentation in this area, and expand upon previous models for axonal NF dynamics. Finally, we address how C-terminal phosphorylation is regionally and temporally regulated by a balance of kinase and phosphatase activities, and how misregulation of this balance can contribute to motor neuron disease.     

Relationship between phosphorylation and synthesis of goldfish optic nerve proteins during regeneration

After intraocular injection of radiolabeled phosphate and 3H-proline, the labeling of goldfish optic nerve proteins was monitored over a 7 week period of regeneration following a lesion to the optic tract. Labeled phosphate incorporation into total nerve protein increased to a peak value about twice that in normal nerve at 3 weeks after injury, then declined to slightly above normal by 7 weeks. Incorporation of 3H-proline showed a higher rise and a steeper decline, with values still significantly above normal at 7 weeks. Two-dimensional gel electrophoresis revealed that almost all the individual proteins examined underwent an increase in 3H-proline incorporation with a peak at about 3 weeks. However, only 4 proteins showed an increase in incorporation of 32P correlated with the increase in 3H-proline. The closest correlation was seen for protein 4, the equivalent of the growth-associated protein GAP-43; for the other 3 proteins (15, 31, and 38) 32P incorporation remained elevated even when 3H-proline incorporation had declined. Two other proteins (24e and 48) showed increased 32P incorporation not correlated with 3H-proline changes. Several proteins showed a decrease in 32P incorporation, even though 3H-proline incorporation was increased. For example, the phosphorylation of ON2, a neuronal intermediate filament protein, showed a long-lasting decline, which was already evident at 1 week and had not yet returned to normal by 7 weeks. Other proteins in this group (33, 37, and 46) showed a faster recovery.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deafferentation-induced abnormal neurofilament phosphorylation in red nucleus neurones

Hippocampal deafferentation has been proposed as a pathogenetic mechanism for neurofibrillary tangle (NFT) formation in human mesolimbocortical dementia. We previously developed a rodent model of hippocampal deafferentation involving bilateral destructive lesions of the ventrotegmental area (VTA), septum of the medial forebrain and entorhinal cortex combined with pharmacological inhibition of serotonin 5-HT2 and dopamine D1 receptors. Unexpectedly, we observed an alteration in phosphorylated neurofilament protein immunoreactivity and argyrophilia in magnocellular neurones of the red nucleus. Here, we determined the neuroanatomical, pharmacological and temporal requirements for this effect on red nucleus neurones. We found that abnormal phosphorylation and argyrophilia were critically dependent on bilateral destruction of the VTA and antagonism of 5-HT2 receptors. Although extensive neurofilament hyperphosphorylation and argyrophilia were observed in red nucleus magnocellular neurones within nine days of treatment, no NFTs were formed and these effects were transitory. Resolution of these cytoskeletal abnormalities was accompanied by increased expression of the calcium binding protein, parvalbumin, suggesting that alterations in intraneuronal calcium levels may modify the deafferentation response.     

Perikaryal neurofilament phosphorylation in axotomized and 6-OH-dopamine-lesioned CNS neurons

The axon reaction in the central nervous system was studied using a monoclonal antibody to phosphorylated neurofilaments. Axotomy was performed by cutting the nigrostriatal pathway. We were able to show that phosphorylated epitopes of neurofilaments, that are usually restricted to axons, could be detected in the perikarya and dendrites of axotomized neurons as early as 3 days postlesion. These neurons remained labelled up to 17 days after axotomy and in some cases even up to 6 weeks. The cytoplasmic changes appearing in the lesioned neurons 8 days after axotomy seem to indicate that these neurons will probably degenerate or survive only in an atrophied, non-functional state as they are unable to regenerate their sectioned axon. Neurochemical lesions, using the neurotoxin 6-OH-dopamine, were performed to establish whether this reaction of perikaryal neurofilament phosphorylation may be a non-specific phenomenon accompanying neuronal degeneration or injury. Although cell loss was important, no labelled neurons could be observed following 6-OH-dopamine treatment. These results indicate that the induction of perikaryal neurofilament phosphorylation is a response to selective types of neuronal injury and concerns selective neuronal populations.     

Early or late appearance of "dropped head syndrome" in amyotrophic lateral sclerosis

BACKGROUND: "Dropped head syndrome" caused by neck extensor weakness has been reported in a variety of neuromuscular disorders. Previously published reports include isolated cases with amyotrophic lateral sclerosis (ALS). In this report, nine patients with ALS and dropped head syndrome seen during a 20 year period are described. PATIENTS AND INVESTIGATIONS: Between 1981 and 2000, 683 patients with ALS were diagnosed, based on El Escorial criteria. Nine of these had profound neck extensor weakness observed as an early feature, or developing during the later stages of the disease. The protocol for evaluation included detailed clinical history, neurological examination, electromyography, and nerve conduction studies. Investigations were undertaken to exclude malignancy, lymphoproliferative disorders, thyroid dysfunction, and collagen vascular disease. RESULTS: The incidence of dropped head syndrome was 1.3%. The mean (SD) age of the affected patients was 53.3 (10.3) years (range 33 to 65), with an equal distribution of cases in the fourth to seventh decades. In six patients, head drop was an early feature (mean interval from onset of illness 11.6 months (range 3 to 24)); in three it was late (between three and eight years after onset). In five patients, mild neck flexor weakness was present in addition to severe extensor weakness. In all nine patients there were diffuse upper and lower motor neurone signs. None of the patients had difficulty in breathing but all had difficulty in swallowing and social embarrassment, both of which could be corrected by simple measures. CONCLUSIONS: Dropped head syndrome is an important clinical sign and usually occurs as an early feature within the first one to two years after the onset of ALS. The cause of dropped head syndrome in these nine cases could be easily established as ALS by the presence of generalised signs.     

Cyclin-dependent kinase 5 increases perikaryal neurofilament phosphorylation and inhibits neurofilament axonal transport in response to oxidative stress

Cyclin-dependent kinase 5 (cdk5) phosphorylates the high molecular weight neurofilament (NF) protein. Overexpression of cdk5 inhibits NF axonal transport and induces perikaryal accumulation of disordered phospho-NF cables. Experimental and clinical motor neuron disease is characterized by oxidative stress, increased cdk5 activity, and accumulation of phospho-NFs within perikarya or proximal axons. Because oxidative stress increases cdk5 activity in experimental motor neuron disease, we examined whether oxidative stress induced cdk5-mediated NF phosphorylation. Treatment of cultured neuronal cells with hydrogen peroxide inhibited axonal transport of green fluorescent protein-tagged NF subunits and induced perikaryal accumulation of NF phosphoepitopes normally confined to axons. These effects were prevented by treatment with the cdk5 inhibitor roscovitine or transfection with a construct expressing the endogenous cdk5 inhibitor peptide. These findings indicate that oxidative stress can compromise NF dynamics via hyperactivation of cdk5 and suggest that antioxidants may alleviate multiple aspects of neuropathology in motor neuron disease.     

Rejection and regeneration through peripheral nerve allografts: immunoperoxidase studies with laminin, S100 protein and neurofilament antisera

The pattern and temporal sequence of histopathological events in a rat nerve allograft model were evaluated. Following grafting and varying survival periods (from 1 to 30 weeks), the host and donor nerve were removed and assessed by light and electron microscopy. Nerve allografts underwent Wallerian degeneration and rejection. Wallerian degeneration was the dominant pathologic process at weeks 1 and 2 after engraftment. Histologic rejection started as an epineurial process at weeks I and 2, became progressively endoneurial and was most prominent at 4 and 6 weeks after engraftment. Rejection was accompanied by evidence of graft Schwann cell and endoneurial tube loss. The rejection process delayed, but did not prevent, nerve regeneration by the host. Regeneration of fine neurofilament-positive axonal sprouts into the proximal portions of the graft was observed as early as week 2. Subsequently, regeneration occurred through the periphery and around the exterior of the rejected nerve allograft fascicle. Regenerating axons were accompanied by S100 protein reactive Schwann cells and newly synthesized laminin-positive endoneurial tubes. Regenerating axons reinnervated the distal host segment at week 8 and increased in number and myelination thereafter. The observations of rejection and regeneration through nerve allograft segments are discussed in reference to previous studies.     

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. I. Production following colchicine application to the sciatic nerve.

Neurofilament (NF) triplet proteins are normally poorly phosphorylated in neuronal perikarya, the two high molecular weight polypeptides becoming extensively phosphorylated once the NF enters the axon. Abnormal expression of phosphorylated NF (pNF) epitopes in neuronal perikarya has been revealed using monoclonal antibodies in a variety of human and experimental conditions. In the present study, we asked whether pNF epitopes are expressed in sensory neurons in the L4 and L5 dorsal root ganglia (DRG) following blockade of fast axonal transport in a model producing few (less than 1%) degenerating fibers. Colchicine (5 mM) was briefly (45 minutes) applied to the sciatic nerve at mid-thigh twice (once weekly) and the animals studied two weeks following the first colchicine application; contralateral nerves were either treated with saline or crushed. Modest to intense immunoreactivity was found with antibody 07-05 (directed against pNF epitopes on the two high molecular weight NF polypeptides) in 30.4% and 45.1% of DRG neurons from colchicine-treated and crushed nerves, respectively; only a rare cell body demonstrated immunostaining from the contralateral saline-treated nerves. Immunoreactivity was not observed with antibody 07-05 at two and five days following single colchicine application. In a separate study, colchicine or saline was applied (as above) 1-2 cm proximal to a nerve crush. Colchicine application did not influence the extent of DRG neurons expressing pNF epitopes; immunostaining with antibody 07-05 was present in 44.7% and 43.8% of DRG neurons from saline-treated and colchicine-treated crushed nerves, respectively. The results indicate that structural interruption of nerve-target contact is not necessary to induce aberrant NF phosphorylation in neuronal perikarya. It is suggested that loss of a retrogradely transported "trophic" signal(s) triggers this response.     

Accumulation of neurofilament proteins in the regenerating facial nerve

In order to compare the extent of axonal regeneration in two surgical nerve repair procedures, we measured the levels of the neurofilament (NF) proteins in the regenerating facial nerve of adult New Zealand rabbits. The animals were operated on bilaterally, with a chamber model placed on one side and a cable graft model inserted on the contralateral side. Normal nerve from unoperated animals or nerve removed during nerve repair surgery served as controls. Using immunoblot techniques and densitometric measurement, we examined specific changes in the individual NF [High (H), Medium (M), and Low (L) molecular weight (MW)] in the regenerating nerve at 3 and 7 weeks postoperation time. Linearity of the densitometric system was established by separation of serial dilutions of known NF on the gel, and blotting for immunostaining. The amount of all 3 NF's decreased during the regeneration process compared to normal nerve, but there were differences between the two procedures. The NFH in the distal segment of the chamber repaired nerve at 7 weeks was 60-70% of the preoperative state, which correlated with a previous morphological study of axona) caliber during regeneration. At 3 weeks, NF content was lower in the distal segment of the chamber than in the distal cable graft. By 7 weeks, NF content was similar in proximal and distal segments of both models. Thus, although initial regeneration is slower in the chamber model, the eventual outcome is similar in both chamber and graft.     

Interleukin-1 promotes expression and phosphorylation of neurofilament and tau proteins in vivo

Slow-release, IL-1-impregnated pellets implanted in rat cerebral cortex to simulate chronic overexpression of IL-1 significantly increased relative tissue levels of tau mRNA and of tau immunoreactivity in neuronal cell bodies and in swollen dystrophic neurites that also overexpressed phosphorylated and nonphosphorylated neurofilament epitopes. In addition, rats with IL-1-impregnated pellets, but not control rats or those with sham pellets, showed focal immunoreactivity for hyperphosphorylated tau epitopes present in paired helical filaments. Our results are consistent with an important driving role for IL-1 in the pathogenesis of Alzheimer-type neuronal and neuritic changes.     

C-terminal phosphorylation of the high molecular weight neurofilament subunit correlates with decreased neurofilament axonal transport velocity

We probed the relationship of NF axonal transport of neurofilaments (NFs) to their phosphorylation state by comparing these parameters in two closely-aged groups of young adult mice - 2 and 5 months of age. This particular time interval was selected since prior studies demonstrate that optic axons have already completed axonal caliber expansion and attained adult NF levels by 2 months but, as shown herein, continue to increase NF-H C-terminal phosphorylation. NF axonal transport was monitored by autoradiographic analysis of the distribution of radiolabeled subunits immunoprecipitated from optic axon segments at intervals following intravitreal injection of 35S-methionine. Both the peak and front of radiolabeled NFs translocated faster in 2- vs. 5-month-old mice. This developmental decline in NF transport rate was not due to reduced incorporation of NFs into the cytoskeleton, nor to an overall decline in slow axonal transport. By excluding or minimizing other factors, these findings support previous conclusions that C-terminal NF phosphorylation regulates NF axonal transport.     

The cerebral cortical appearance in late paraphrenia

The cortical appearance on CT scan in patients suffering from late paraphrenia was assessed compared to that of age-matched normal controls. There was no difference in overall cortical appearance between the two groups. However, a correlation existed between ventricular size and cortical size and between ventricular size and age in normal controls but this was not present in paraphrenia. The implications of these findings are discussed in relation to a possible structural abnormality in late paraphrenia.