Neurofilaments assume a less random architecture at nodes and in other regions of axonal compression

Neurofilament distributions were mathematically characterized in four chicken somatic motor axons at each of four histologically distinct regions: compact myelinated regions, compact myelinated regions associated with Schwann cell nuclei, Schmidt-Lanterman clefts, and nodes of Ranvier. Compact myelinated regions had the largest cross-sectional areas, the lowest neurofilament densities, and the most random neurofilament organizations — nodes of Ranvier had the smallest cross-sectional areas, the highest neurofilament densities, and the most ordered architectures. In these myelinated axons, the closest natural neurofilament spacing was 25 nm. Mathematical analyses of serial sections suggested that neurofilament interactions are sufficiently weak and transient to permit a full range of variation from random to ordered cytoskeletal architectures as the neurofilaments move longitudinally through the few micron span of the paranodal-nodal region of a single axon.     

Ultrastructural studies of the superior cervical trunk of the mouse: distribution, cytochemistry and stability of fibrous elements in preganglionic fibers.

The ultrastructure of axons in the preganglionic cervical sympathetic trunk of the mouse is described with emphasis on the number, distribution and stability of fibrous elements in the axoplasm. Neurofilaments outnumbered microtubules in myelinated and non-myelinated axons of all sizes, and the ratio of neurofilaments to microtubules in non-myelinated axons at each point studied was fairly consistent and independent of axonal diameter. The density of neurofilaments and microtubules, however, was greater in axons of progressively smaller diameter. In non-myelinated axons and small myelinated axons neurofilaments were uniformly distributed throughout the axoplasm resulting in minimum and maximum interfilament distances of 300 angstrom and 500 angstrom respectively; the spacing of fibrous elements within any one axon was dependent upon its diameter and position with respect to the superior cervical ganglion in the preganglionic trunk. The maximum interfilament distance was also found in large myelinated axons where neurofilaments, occurring in fascicles, were separated by distances of approximately 500 angstroms. Cytochemical staining of axons with lanthanum hydroxide, ruthenium red or alkaline bismuth delineated the delicate filamentous matrix interconnecting microtubules, neurofilaments and other organelles in the axoplasm. Alkaline bismuth stain was most intense in myelinated axons where heaviest deposition of reaction product was associated with neurofilaments. Treatment in vitro of the cervical sympathetic trunk with 5 X 10(-5) M vinblastine sulfate dissociated microtubules and induced formation of crystalline arrays of "tubular" elements. A uniform center to center spacing of 250-300 angstrom was found for crystalloids in non-myelinted axons; however, in myelinated axons the center to center spacing was not uniform and varied in the range 300-600 angstrom. Neurofilaments and their surface projections were unaffected by vinblastine. Fixation in the presence of lanthanum enhanced delineation of crystalloid elements. Exposure of 0-4 degrees C for up to three hours had no consistent effect on microtubules or neurofilaments. In contrast, cold treatment disrupted the delicate axonal matrix and resulted in the formation of aggregates of coarse flocculent material in the axoplasm.     

Abnormal expression of neurofilament proteins in dysmyelinating axons located in the central nervous system of jimpy mutant mice

Myelination in the peripheral nervous system is considered to increase the phosphorylation level of neurofilament proteins in the axon, resulting in an increase in axonal calibre. To understand the relationship between myelination and neurofilament proteins in axons, we examined jimpy mutant mice with a point mutation in the proteolipid protein gene and dysmyelination in the central nervous system. The jimpy mice exhibited a characteristic similarity in neurofilament nature to the myelin-deficient mice in the peripheral nervous system reported previously. The following novel results were obtained in the jimpy mice: dysmyelinated axons, in which the amount of non-phosphorylated neurofilament-H was drastically increased without a significant reduction of the phosphorylated form, compared with the control myelinated axons, did not suffer any decrease in their diameters. Expression levels of all neurofilament subunit proteins and their mRNAs were enhanced in the central nervous system tissue. Because the above biochemical data were obtained from the cytoskeletal fraction, at least some of the increased neurofilament-H and -M proteins appeared to be coassembled into neurofilaments but remained non-phosphorylated. Axonal neurofilaments of the jimpy were, probably due to this abnormal stoichiometry and phosphorylation state in neurofilaments, more compact and random in alignment with less prominent cross-bridges than those of the control, providing possible evidence for disturbing the axonal transport of other organelles. These results suggest that myelination regulates both the expression and phosphorylation of neurofilament proteins, and is essential for the cytoplasmic organization of myelinated axons.     

Microtubules have special physical associations with smooth endoplasmic reticula and mitochondria in axons

Ultrastructural morphometry was used to document the non-random spatial distributions of organelles within the compact myelinated region of avian oculomotor axons. These regions contain large numbers of loosely packed neurofilaments (NFs) (241/μm²) and only a relatively small number of microtubules (MTs) (4/μm²), mitochondria (0.6/μm²), and smooth endoplasmic reticulum (SER) (1.6/μm²). Random co-occurrences between the relatively sparsely distributed MTs, mitochondria, and SER are probably infrequent in these axons. The actual co-occurrences of MTs, mitochondria, and SER with MTs were counted and compared to the co-occurrences expected in a random Poisson distribution. At long distances (200 nm), the co-occurrences were random. At shorter distances (40 nm and less), MTs were still randomly associated with other MTs. However, at these shorter distances, the spatial associations of mitochondria with MTs and of SER with MTs were not random; such preferential stable associations may be produced by specific MT associated cross-bridging proteins. In axons, MTs tend to be clustered together, giving the appearance of MT bundles. We propose that the MT-MT bundling is an indirect result of MT concentration along the continuous intra-axonal SER network, to which the MTs are apparently tied directly by dynamic molecular cross-bridges.     

Decreased axon caliber and neurofilaments in hereditary motor and sensory neuropathy, type I

The axons of large- and intermediate-diameter myelinated fibers of sural nerves of patients with hereditary motor and sensory neuropathy, type I (HMSN-I), were previously found to be attenuated relative to their myelin spiral length. We inferred that axonal atrophy might account for secondary segmental demyelination and remyelination. To assess whether the observed axonal atrophy could be explained by a decrease in neurofilaments, we have evaluated the number of neurofilaments, microtubules, and other axon organelles in sural nerves of patients with HMSN-I. Whereas the density per square micrometer of neurofilaments or microtubules in diseased nerves was not significantly different from that in control specimens, the number of neurofilaments per axon as related to myelin spiral length was significantly less for intermediate and large myelinated fibers in HMSN-I nerves. The regression lines for the number of microtubules per axon on myelin spiral lengths were also less steep in HMSN-I, but the difference did not reach statistical significance. These results indicate that the number of neurofilaments is proportional to axon diameter but significantly below that expected considering myelin spiral length. Decreased neurofilament synthesis, assembly, or transport may underlie the axonal atrophy in HMSN-I.     

Unmyelinated fibers in the pyramidal tract of the rat: a new view

An electron microscopical morphometric analysis has been carried out at the medullary level of the pyramidal tract of the rat in order to quantify both myelinated and unmyelinated fibers: it was found that unmyelinated fibers outnumber myelinated ones substantially (133,000 ± 18,000 versus 91,000 ± 11,000, respectively). The unmyelinated fibers range from 0.05 to 1 μm in a monomodal distribution (mean 0.16 μm). For myelinated axons also a monomodal distribution was observed (ranges: axon 0.10–4 μm, mean 0.80 μm; myelin sheath 0.25–5 μm, mean 1.19 μm).     

Loss of neurofilaments in the neuromuscular junction in a rat model of proximal axonopathy

C. Soler‐Martín, Ú. Vilardosa, S. Saldaña‐Ruíz, N. Garcia and J. Llorens (2012) Neuropathology and Applied Neurobiology 38, 61–71 Loss of neurofilaments in the neuromuscular junction in a rat model of proximal axonopathy Aims: Rodents exposed to 3,3′‐iminodipropionitrile (IDPN) develop an axonopathy similar to that observed in amyotrophic lateral sclerosis motor neurones, in which neurofilaments accumulate in swollen proximal axon segments. This study addressed the hypotheses that this proximal axonopathy is associated with loss of neurofilament proteins in the neuromuscular junctions and a progressive loss of neurofilaments advancing in a distal‐proximal direction from the distal motor nerve. Methods: Adult male Long‐Evans rats were exposed to 0 or 15 mM of IDPN in drinking water for 1, 3 or 5 weeks, and their distal axons and neuromuscular junction organization studied by immunohistochemistry. Quantitative data were obtained by confocal microscopy on whole mounts of the Levator auris longus. Results: Muscles showed no change in the distribution of acetylcholine receptor labelling in the neuromuscular junctions after IDPN. In contrast, the amount of neurofilament labelling in the junctions was significantly reduced by IDPN, assessed with two different anti‐neurofilament antibodies. In preterminal axons and in more proximal axon levels, no statistically significant reductions in neurofilament content were observed. Conclusions: The proximal neurofilamentous axonopathy induced by IDPN is associated with an abnormally low content of neurofilaments in the motor terminals, with a potential impact in the function or stability of the neuromuscular junction. In contrast, neurofilaments are significantly maintained in the distal axon.     

Slowing of neurofilament transport and the radial growth of developing nerve fibers

Several lines of evidence indicate that neurofilaments are major intrinsic determinants of axonal caliber in myelinated nerve fibers, and that the delivery of neurofilaments by slow axonal transport is an important mechanism by which neurons regulate axonal caliber. To further clarify the relationship between neurofilament transport and axonal caliber, we examined transport in developing motor fibers of rat sciatic nerve. In 3-, 10-, 12-, and 20-week-old rats, lumbar motor neurons were labeled by the intraspinal injection of radioactive amino acids, and the distributions of labeled cytoskeletal proteins within the sciatic nerve were analyzed at various times afterwards using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel fluorography, and liquid scintillation spectroscopy. There was a progressive decline in the velocity of neurofilament transport with increasing distance along axons undergoing radial growth. By examining transport in different regions of the nerve in animals of the same age, we separated age-dependent reductions in velocity from those related to position along the nerve. The cross-sectional areas of these motor axons (in the L5 ventral root) increased linearly between 3 and 18 weeks of age. Quantitative electron microscopic analysis at 3 and 10 weeks of age revealed that neurofilament density was comparable in fibers of all calibers, indicating that the radial growth of these myelinated nerve fibers correlates with a proportional increase in neurofilament content. We propose that progressive reduction in the velocity of neurofilament transport along the nerve provides for radial growth during development.     

A useful programme in BASIC for axonal morphometry with introduction of new cytoskeletal parameters.

Interest in the structure of axons and quantification of their components has been growing over the last years. However, the existing literature contains few reports of available computer programmes to facilitate such studies. This paper presents a fully comprehensive BASIC programme for the morphometric analysis of electron micrographs of cross-sectional nerve fibres. From drawings of fibre and axonal contours and dots of the microtubules and neurofilaments, the programme calculates the following parameters: area, diameter and form factor of the fibres and axons, number and density of microtubules and neurofilaments, proportion between microtubules and neurofilaments (R-proportion), myelin thickness and the diameter of the axon relative to its sheath (g-ratio). The programme also introduces three new parameters to analyse the degree of uniformity of microtubule and neurofilament distribution: distances between microtubules and between neurofilaments, equilateral index and cytoskeletal intermingling index. The programme is written in Microsoft BASIC Interpreter for Apple Macintosh (Microsoft Corporation) but can be used on other computers. Although the programme has been tested on adult rat optic nerve fibres, it can be used for different projects concerning axonal morphometry.     

The return of phosphorylated and nonphosphorylated epitopes of neurofilament proteins to the regenerating optic nerve of Xenopus laevis

Neurofilament proteins of mammalian axotomized peripheral axons, which regenerate effectively, resemble those of embryonic axons. However, injured centrally projecting mammalian axons, which fail to regenerate, have very different neurofilament compositions than during development. If changes in neurofilament composition after injury reflect the ability of axotomized neurons to regenerate effectively, then the neurofilaments of centrally projecting axons that can regenerate should more closely resemble those of developing axons. In this study, the neurofilament compositions of injured optic axons of the frog, Xenopus laevis, were examined, since these axons can regenerate a fully functional projection. Antibodies to phosphorylated and nonphosphorylated forms of neurofilament proteins that had been used previously to study the neurofilament composition of newly developing X. laevisoptic axons were used in immunocytochemical studies to examine the return of neurofilaments to the optic nerve after an intraorbital nerve crush. Intraocularly injected wheat germ agglutinin conjugated to horseradish peroxidase was used to label the regenerating axons independently of their neurofilaments. Neurofilament immunoreactivities disappeared rapidly from crushed axons during the first week after surgery. By nine days after surgery, antibodies to nonphosphorylated form of middle (NF-M) and low molecular weight (NF-) neurofilament proteins and the Xenopusneuronal intermediate filament protein (XNIF) began to stain the nerve just beyond the lesion. By this time, however, growing axonal terminals had reached the optic chiasm. Antibodies to phosphorylated epitopes of NF-M began to stain axons at 15 days, just as growing axons began to arrive at the optic tectum. Nonphosphorylated high molecular weight neurofilament protein (NF-H) began to appear in axons between 18 and 21 days after surgery. Thus, the reappearance of neurofilaments during optic axon regeneration resembled the general pattern seen during development. The chief difference between development and regeneration was that neurofilament epitopes took longer to emerge during regeneration. One possibility is that cues encountered along the optic pathway influence the neurofilament composition of retinal ganglion cell axons. Then, the greater distance travelled by regenerating axons could account for the longer time taken for their neurofilament compositions to mature. © 1994 Wiley-Liss, Inc.     

Effect of changes in neurofilament content on caliber of small axons: the beta,beta'-iminodipropionitrile model

The structural role of neurofilaments in the normal axon and the consequences of altered axonal transport of neurofilaments have been extensively studied in large axons. These studies suggest that neurofilament numbers and interneurofilament spacing are major determinants of axonal cross-sectional area. In contrast, in small axons and dendrites, microtubules and membranous organelles appear to be the most closely correlated with size and shape of the cell process. In this study we have examined the effect of impairment in neurofilament transport on small axons, typical of most CNS pathways. Neurofilament transport was impaired by administration of beta,beta'-iminodipropionitrile (IDPN), resulting in proximal accumulation and distal depletion of neurofilaments. The evolution of these changes was studied in the optic nerves of guinea pigs treated with IDPN, 1-35 weeks following intoxication. The effect of this redistribution of neurofilaments on cross-sectional area of small axons was evaluated using quantitative ultrastructural methods. Our results show that with the alteration in neurofilament transport seen with IDPN intoxication, there is a wide spectrum of neurofilament densities, ranging from a 5-fold increase above normal in the proximal axon, to a 5-fold decrease below normal in the distal axon. Although the optic nerve fibers enlarge with the increase in neurofilament content, they do not atrophy significantly with the continued loss of neurofilaments. We conclude that factors other than neurofilament content are capable of maintaining size and shape of these small axons. Candidate organelles include microtubules and membranous organelles and possibly other axonal elements.     

Traumatic compaction of the axonal cytoskeleton induces argyrophilia: histological and theoretical importance

It was earlier established that one of the primary morphopathological consequences of experimental traumatic brain injury is a dramatic reduction in the distances between the neurofilaments (cytoskeletal compaction) inside a number of axon segments that appear to be randomly distributed among normal axons in an otherwise undamaged parenchymal environment. The present results demonstrate that the cytoskeletal compaction instantly induces argyrophilia, thereby rendering possible selective visualisation of the affected axon segments for light microscopy through use of a special silver staining method. On combination of this method with electron microscopy, it was revealed that the cytoskeletal compaction is completed in much shorter times and extends to much longer axon segments than previously assumed.     

Neurofilament sidearm proteolysis is a prominent early effect of axotomy in lamprey giant central neurons

In the accompanying paper, it was shown that axotomy of lamprey spinal axons induces the rapid formation of condensed neurofilamentous masses in the proximal axon stump near the lesion. In this study, we used immunocytochemical and Western blot analysis to characterize these masses further and to determine the time course of their formation and dispersal. We show that monoclonal antibodies specific to the “rod” domain of lamprey neurofilament protein strongly stain such masses in tissue sections without staining other axonal neurofilaments. Antibodies specific for the neurofilament “sidearm” domain fail to recognize neurofilamentous masses but stain other axonal neurofilaments. Western blots of spinal cord segments from the lesion site were compared to unlesioned cord and to samples of cord distant from the lesion. We found that a neurofilament rodrspecific antibody identified breakdown products of the same size as the rod domain in samples from the lesion site, but not elsewhere. Other lesion-specific neurofilament breakdown products were recognized by a sidearm-specific antibody. This lesion-specific pattern of neurofilament proteolysis was visible at 1 day postlesion and was still present 3 weeks later. Immunocytochemistry showed masses of rod-staining neurofilaments in axon stumps by 12 hours postlesion that remained for 1–2 weeks postaxotomy; these dispersed with the onset of regeneration. Such neurofilament rod staining was also prominent in distal axon stumps undergoing Wallerian degeneration. We conclude that axotomy induces neurofilament sidearm proteolysis near the lesion, permitting antibody access to the rod domain. We suggest that sidearm loss causes the high packing density of neurofilaments within neurofilamentous masses near the lesion site and that neurofilament sidearm proteolysis can be used to distinguish degenerative from regenerative changes in lesioned lamprey axons. © 1995 Wiley-Liss, Inc.     

Rapid reorganization of the axonal cytoskeleton induced by a gamma diketone

Microtubules and neurofilaments are the major components of nerve fiber axoplasm and are evenly distributed longitudinally. Rearrangement of this distribution can be induced by a number of chemicals, including 2,5-hexanedione (2,5-HD). Utilizing 2,5-HD, this study explores the earliest time points of observable rearrangement of the cytoskeletal elements. Intrafascicular injections into the sciatic nerve shows central accumulation of microtubules and peripheral relocation of neurofilaments within 1 min of injection, suggesting a direct effect of the toxin of these components.     

Overexpression of neurofilament subunit M accelerates axonal transport of neurofilaments

Neurofilaments are composed of three polypeptide subunits (NF-H, NF-M and NF-L). They are the most abundant cytoskeletal element in large myelinated axons and play a central role in development of axonal caliber. To perform this role, neurofilaments are transported from their site of synthesis, the cell bodies, to the distal axons. Previous studies showed that overexpression of NF-M in transgenic mice led to accumulation of neurofilaments in neurons and a reduction in the number of neurofilaments in axons, suggesting that axonal transport of neurofilaments was slowed. To determine whether this was the case, we measured axonal transport velocities in the wild type and transgenic mice overexpressing NF-M by the classical pulse-labeling method using 35S-methionine. We found that neurofilament transport in peripheral motor axons can be described with a model consistent with two linear velocities. Contrary to expectations, both velocities were accelerated by overexpression of NF-M. These results suggest that subunit composition in neurofilaments play a regulatory role in neurofilament transport. In addition, these results show that there are regional differences in neurofilament transport along long axons and these differences may be the basis for selective regional accumulation of neurofilaments in various neurological disorders.     

A monoclonal antibody to mammalian neurofilament protein stains somata and dendrites in gymnotid fish

Monoclonal antibody N210 (mabN210) recognizes the 210 kdalton neurofilament protein in mammals and gives characteristic immunocytochemical staining of neurofilament-rich processes. For example, in the cerebellum it recognizes myelinated axons and the calyx formed by basket cell axon collaterals. The distribution of mabN210 immunoreactivity was studied in the gymnotid brain (Apteronotus albifrons). In contrast to the mammalian distribution, mabN210 immunoreactivity was not found in most axons of the gymnotid brain. Instead, deposits of reaction product were present in the somata and dendrites of most neurons and were especially dense in those neurons with extensive dendritic trees, the Purkinje cells, pyramidal cells of the electrosensory lateral line lobe, the crest cells of the nucleus medialis and the pyramidal cells of the tectum. Electrosensory lateral line lobe pyramidal cells are known to contain few, if any, neurofilaments in their dendrites. Western blots of whole gymnotid brain proteins demonstrated that mabN210 recognizes two polypeptides apparent molecular weights 60 and 19 kdaltons. These proteins are thus antigenically similar to neurofilament protein and their expression in the gymnotid brain may be related to the peculiar dendritic branching pattern of Purkinje cells and similar cell types.     

Neuropathology of ALS: Spheroids

I briefly review spheroids observed in the anterior horns of the spinal cord in amyotrophic lateral sclerosis (ALS). Spheroids are argentophilic bodies more than 20 μm in diameter. Recently, some connections between the proximal axonal swellings including spheroids and the perikarya have been reported in some ALS patients with a short clinical course or mild depletion of anterior horn neurons. Most of the cell bodies directly connected with the axonal swellings appear normal, and spheroids are considered to be one of the hallmarks of the early histological changes in this disorder. Spheroids are strongly positive with anti-phosphorylated neurofilament antibody, and are also positive with calcitonin gene-related peptide and anti-peripherin antibody. Some spheroids are immunostained with anti-synaptophysin antibody and anti-ubiquitin antibody. Spheroids are not immunostained with anti-phosphorylated tau antibody, or high molecular weight microtubule associated proteins. Electron microscopically, spheroids are usually composed of densely packed accumulation of 10 nm neurofilaments with a variety of orientations, plus vesicles, dense bodies and mitochondria. When the swellings of the initial segment is relatively pronounced, the undercoating is obscured and the neurofilaments become interwoven in some parts. In the first internode of the myelinated axons, as the swellings become larger, the neurofilaments lose their parallel orientation and become intermingled. Large accumulation of neurofilaments resembling spheroids in the perikarya of large anterior horn cells suggests that spheroids could be derived not only from the axon including the proximal portion, but also from the perikarya. Structures apparently identical to axonal spheroids are observed at the light and electron microscopic levels in the proximal portion of axons of anterior horn cells in animal models intoxicated with β, β'-iminodipropionitrile (IDPN), or with aluminum, in hereditary canine spinal muscular atrophy (HCSMA). The pathogenetic mechanism is probably associated with an impairment in slow axonal transport which particularly affects the neurofilaments in IDPN and aluminum intoxication. Impairment of slow axonal transport of neurofilaments also plays an important role in the pathogenesis of ALS. The average diameter of even normalappearing initial segment is larger in ALS than in the controls. The perikarya connected with the swollen proximal axons and their dendrites almost always appear normal. These findings suggest that the slow axonal transport of neurofilaments is probably impaired in this portion of the axon at an early stage in ALS as well as animal models for human ALS. However, techniques to analyze slow axonal transport in humans still remain tobe developed. Recently, overexpression of neurofilament subunits in transgenic mice produces a condition resembling ALS. The transgenic model may offer an interesting perspective not only for testing therapeutic strategies but also for investigating in a systematic way the various genetic and environment factors controlling the onset and progression of the disease and might yield new insights on the etiology of ALS.     

Regional variation in the abundance of axonal cytoskeletal proteins.

The relative abundance of several axonal cytoskeletal proteins was determined by immunoassay at various sites in the peripheral and central nervous systems of adult rats. Within the peripheral nervous system, the ratio of tubulin to neurofilaments was greatest for nerves composed of unmyelinated axons and least for nerves with large myelinated axons. MAP1 protein was more prominent in unmyelinated fibers; conversely tau proteins were relatively more abundant in large myelinated axons. An immunochemical index of neurofilament phosphorylation was less for unmyelinated fibers than for myelinated ones. In the fimbria-fornix, pyramidal tract, and superior cerebellar peduncle, similar trends were observed: small axons had more MAP1, less tau, and a greater ratio of tubulin to neurofilament proteins. The phosphorylation index was greatest for the superior cerebellar peduncle, the tract with the largest axons. The immunochemical index of neurofilament phosphorylation was greater for the optic nerve than for axonal tracts in the brain proper. These results suggest that development of large myelinated axons is associated with greater neurofilament content, neurofilament phosphorylation, and with greater abundance of tau proteins in the CNS and the PNS; however, quantitative aspects of these relationships differ in the PNS and the CNS.     

Characterization of the calcium-induced disruption of neurofilaments in rat peripheral nerve

Transverse frozen sections of desheathed rat peripheral nerve were incubated in media of different composition prior to fixation and processing for electron microscopic examination. Neurofilaments remained intact when these tissues were incubated in calcium-free media. A loss of neurofilaments and their replacement by granular debris occurred in myelinated and unmyelinated fibers following incubation in media containing 2 mM calcium. The calcium-mediated disruption of neurofilaments was inhibited by preincubation or incubation with 1 mM p-chloromercuribenzoate (PCMB). The inhibition by preincubation with PCMB could be partially reversed by subsequent preincubation with 10 mM dithioerythritol (DTE). Calcium-mediated breakdown of neurofilaments did not occur after prolonged preincubation in calcium-free media, a finding which suggested that neurofilament disruption was dependent upon a tissue factor which could be lost or inactivated in frozen-sectioned nerve tissues. The findings of the present study provide morphological evidence that neurofilament disruption in mammalian peripheral nerve is mediated by a calcium-activated, PCMB-sensitive enzyme in the axoplasm of myelinated and unmyelinated nerve fibers.     

Immunocytochemical and ultrastructural studies of neuronal and oligodendroglial cytoplasmic inclusions in multiple system atrophy

Summary Neuronal alterations in five cases of multiple system atrophy (MSA) were investigated histologically, immunocytochemically and ultrastructurally. Argentophilic neuronal cytoplasmic inclusions (NCIs) were observed in all cases. They were distributed, in order of decreasing frequency, in the pontine nucleus, striatum, subiculum, amygdala, hippocampus, dentate fascia, substantia nigra and inferior olivary nucleus. Anti-ubiquitin antibodies visualized many thickened neurites in the degenerating gray matter as well as NCIs. Some NCIs were also recognized by anti-phosphorylated neurofilament antibodies. Ultrastructurally, NCIs consisted of a meshwork of granule-associated filaments, the diameter ranging from 18 to 28 nm, that were mixed with neurofilaments. The granule-associated filaments were also present in the axoplasm of myelinated fibers. Our studies demonstrate widespread distribution of NCIs in the central nervous system of MSA. The same pathological process that forms the granule-associated filaments in axons may also be responsible for the formation of ubiquitin-positive thickened neurites. These axonal alterations, as well as neuronal perikaryal changes, may play an important role in the impaired neuronal function in MSA.A part of this study was presented at the 67th Annual Meeting of the American Association of Neuropathologists (Baltimore, Md., June 19–22, 1991) with the abstract in J Neuropathol Exp Neurol (1991) 50:307