Axonal maturaion in development—I. Characterization of monoclonal antibodies reacting with axon-specific neurofilament epitopes

Monoclonal antibodies reacting with the high molecular weight neurofilament polypeptides (NF 150K and NF 200K) were obtained upon immunization with NF 150K and NF 200K isolated from bovine spinal cord by anion exchange chromatography. The five monoclonal antibodies obtained with NF 200K stained only axons. With three monoclonals the reactivity was abolished by digestion with phosphatase and by dilution of the supernatants in sodium potassium phosphate. The nine monoclonal antibodies obtained upon immunization with NF 150K stained both high molecular weight neurofilament polypeptides on immunoblots of bovine and rat spinal cord extracts with the exception of one monoclonal only reacting with the homologous antigen. The antibodies could be divided into two groups, axon-specific and conventional. Conventional antibodies decorated neurofilaments regardless of their location, i.e. axons, perikarya and dendrites. With all these antibodies the immunostaining was not affected by phosphatase digestion of neurofilament protein nor by dilution of the supernatants in sodium potassium phosphate. Axon-specific antibodies reacting with both NF 150K and NF 200K in rat spinal cord only stained the heterologous antigen (NF 200K) in rat optic nerve and sciatic nerve extracts. We suggest that some axon-specific neurofilament antibodies recognize neurofilament modifications other than phosphorylation; or, alternatively that they react with phosphorylated epitopes not accessible to phosphate or to exogenous phosphatases. Furthermore, we suggest that some neurofilament modifications do not occur uniformly throughout the nervous system.     

Neurofilament phosphorylation in the axonless horizontal cells of rat retina

Axonless horizontal cells in the outer plexiform layer of rat retina were studied with 19 monoclonal antibodies reacting with phosphorylated and non-phosphorylated epitopes of the two high molecular weight neurofilament proteins (NF 150K and NF 200K). With 6 antibodies, immunoreactivity was confined to the nerve fiber layer on the inner surface of the retina. Horizontal cells were not stained. Four antibodies in this group were axon-specific, while the remaining two stained motor and sensory neuron perikarya in rat spinal cord and dorsal root ganglia, respectively. Of the 13 antibodies which stained horizontal cells, 11 reacted with phosphorylated epitopes and failed to decorate motor neuron perikarya in the spinal cord, while in dorsal root ganglia, they stained a subpopulation of sensory neurons.     

Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.     

Maturation of a large neurofilament protein (NF 150K) in rat postnatal development

The mammalian neurofilament is made of three neuron-specific proteins with approximate molecular weights of 70 kilodaltons (kDa) (NF 70K), 150 kDa (NF 150K), and 200 kDa (NF 200K) by SDS-PAGE. As previously reported in the rat by Strocchi et al (J Neurochem 39:1132-1141, 1982) and Nixon et al (J Cell Biol 94:150-58, 1982), NF 150K comprises three molecular weight variants with the same isoelectric point. A fourth lower molecular weight and slightly less acidic variant was identified by monoclonal and polyclonal antibodies reacting with the alpha-helical middle domain of NF 150K. With few exceptions, this lower molecular weight variant did not stain with monoclonal antibodies reacting with the peripheral carboxy-terminal domain. Staining with these antibodies was abolished or markedly reduced following neurofilament dephosphorylation. The distribution of the NF 150K variants varied in different regions of the nervous system. The higher molecular weight variant (component a) was less prominent in brain compared to spinal cord, optic nerve, and sciatic nerve. Furthermore, the lower molecular weight variant (component d) was not identified in optic nerve and sciatic nerve. All four variants were identified in brain and spinal cord extracts of newborn rats with monoclonal and polyclonal antibodies reacting with the alpha-helical middle domain of NF 150K. As a general (see Results for exceptions) monoclonal antibodies reacting with the carboxy-terminal region of NF 150K did not stain the variants in newborn rat brain extracts until day 10 when immunoreactivity of component a first appeared. The adult pattern was first observed on postnatal day 15.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

Novel monoclonal antibodies provide evidence for the in situ existence of a nonphosphorylated form of the largest neurofilament subunit

We have obtained five monoclonal antibodies to the Mr 200,000 neurofilament component (NF200) after immunization with polypeptides purified from enzymatically dephosphorylated bovine neurofilaments. In immunoblots of untreated neurofilament protein and protein from filaments exposed to phosphatase, these antibodies recognize nonphosphorylated or dephosphorylated, but not phosphorylated, forms of NF200. The epitopes recognized by these new monoclonal antibodies reside in the carboxyterminal domain of the NF200 polypeptide as defined by immunoreaction with limited chymotryptic fragments. Immunohistochemical studies of bovine cerebellum, spinal cord, trigeminal ganglion, and trigeminal nerve with these new monoclonal antibodies demonstrate immunoreactivity primarily in neuronal perikarya; axons and dendrites are weakly or infrequently immunostained. After enzymatic dephosphorylation of these tissues, a more extensive distribution of immunoreactivity is seen, especially in axons and dendrites. Immunostaining of cultured rat sympathetic neurons is restricted to cell bodies. These data provide evidence for the in situ existence of NF200 epitopes that are not phosphorylated in some classes of neurons or regions of a neuron, but are modified by phosphorylation in other neurons or neuronal domains. These new monoclonal antibodies are distinctly different from those in a large library (over 100) raised to, and specific for, phosphorylated neurofilament proteins. They are novel tools for probing neurofilament distribution, metabolism, structure, and possibly function.     

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.     

Axonal regeneration in old multiple sclerosis plaques

Summary Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225–232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.e., after regenerating axons had reached the distal stump of the transected sciatic nerve. Both multiple sclerosis plaques were positive for GFAP and vimentin, but negative for GHAP, while astrocytes in myelinated spinal cord white matter stained with both GFAP and GHAP antibodies. Laminin immunoreactivity in the plaques and normal spinal cord was confined to blood vessels. One plaque was almost devoid of axons as evidenced by indirect immunofluorescence with neurofilament antibodies. Another plaque was packed with bundles of thin axons running an irregular course in the densely gliosed tissue. Axons in the plaque only stained with neurofilament antibodies reacting with sciatic nerve in the early stages of regeneration while axons in the surrounding myelinated white matter were decorated by all neurofilament antibodies, regardless of the time of appearance of immunoreactivity in crushed sciatic nerve. It is concluded that reactive astrocytes forming glial scars do not constitute a non-permissible substrate for axonal growth.Supported by NIH grant NS 13034 and by the Veterans Administration     

Early and late appearance of neurofilament phosphorylation events in nerve regeneration

Neurofilament phosphorylation in regenerating rat sciatic nerve was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes of the 2 large polypeptides of the neurofilament protein triplet (NF 150K, NF 200K). One group of antibodies decorated axons early in the process. In fact, no differences were seen in double labeled sections between these antibodies and polyclonal neurofilament antibodies as to their reactivity with the distal stump of transected sciatic nerves. Another group stained axons after they had completed their elongation, i.e., after they had reached the distal part of the denervated sciatic nerve. In general, the epitopes recognized by antibodies in this group appeared more sensitive to phosphatase digestion as compared to the first group. Furthermore, there was a good correlation between the thickness of the regenerated axons and staining with these monoclonal antibodies. Thick axons (like those observed in normal nerves) were stained, while bundles of thin axons remained unstained. Monoclonal II32 stained regenerated axons in a remarkable segmental pattern. With this antibody, continuous decoration of the axons was still not observed 7 weeks after transection, the longest follow-up period in this study. We suggest that some neurofilament phosphorylation events may contribute to the stabilization of the axonal cytoskeleton and that abnormalities persist in regenerated axons as to the extent of neurofilament phosphorylation.     

Structural similarities and differences between neurofilament proteins from five different species as revealed using monoclonal antibodies

Neurofilament (NF) proteins isolated from human, rabbit, rat, and chicken spinal cord white matter were immunoblotted with monoclonal antibodies (MAbs) raised to bovine NF immunogens. The aim of these experiments was to test the degree of epitope conservation among NF proteins from different vertebrate species. In so doing, the validity of using antibodies raised to NF from one species (cow) for detecting NF proteins of other species was also tested. The MAbs used for these experiments were characterized using bovine NFs to define polypeptide specificity, the approximate location of epitopes within each NF polypeptide, and the effects on antibody recognition of the presence or absence of phosphorylated residues in these polypeptides (see Carden et al., 1985). Our findings indicate that epitopes located in the alpha-helical core domains of NF-H, NF-M, and NF-L are distinct from each other, yet are strongly conserved among the different species. Epitopes located in the noncore or peripheral domains of the NF polypeptides show variable degrees of cross-species preservation. For example, all epitopes in the peripheral domains of bovine NF-H that require the presence of phosphate groups for recognition (phosphorylation dependent) are widely expressed in all species studied. Many phosphorylation-dependent epitopes of bovine NF-H are also present in bovine NF-M, as well as in NF-H of non-bovine species. In addition, epitopes in the peripheral domain of NF-H that require dephosphorylation of NF polypeptides for recognition (dephosphorylation dependent) are also found in NF-H of other mammalian species but not in NF-H of chicken.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.     

Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments

The phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye ("Stains-all") binding to NF polypeptides resolved on SDS-polyacrylamide gels. Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively. Incubation of NF preparations with E. coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L. This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms. "Stains-all" was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins. Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes. Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively. NF-H bound the dye less efficiently than was expected from its phosphate content. After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with "Stains-all," the staining intensity of NF-M decreased by 20-40%, and the staining of NF-L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Formation of 10-nanometer filaments from the 150K-dalton neurofilament protein in vitro

In the present study we report self-assembly of individual neurofilament (NF) triplet proteins (70K, 150K, and 200K daltons) isolated by anion exchange chromatography from bovine spinal cord. Formation of smooth 10-nm filaments by both NF 150K and NF 70K is shown. Optimal conditions for NK 150K filament formation were incubation in 100 mM MES, 0.2 M NaCl, 1 mM DTT, 0.5 mM EGTA, pH 6.5, at 37 degrees C for 24 hr. Under the same assembly conditions, NF 200K formed 7-nm coiled structures. These thin filaments were similar to those formed by NF 70K and 150K under less than optimal conditions. Our results indicate that NF 150K is an integral part of the filament (self-assembly of NF 70K was previously demonstrated by others). We suggest that the optimal conditions resulting in the formation of a 10-nm 200K homopolymer remain to be determined and that the thin coiled structures formed by all three NF proteins are protofilaments that coalesce to form a double helical 10-nm filament.     

Studies of neurofilaments that accumulate in proximal axons of rats intoxicated with β,β′-iminodipropionitrile (IDPN)

The paradigm of IDPN neuropathy was produced in rats in order to examine the neurofilaments (NFs) that accumulate in the proximal motor and sensory axons of intoxicated animals, and to compare the aggregated NFs with control NFs and with the depleted populations of NFs in the distal portions of the same experimental nerves. NFs were probed biochemically and histochemically, using a large and well-characterized library of monoclonal antibodies that included antibodies that are monospecific for each of the rat NF protein subunits (NF-H, NF-M, and NF-L) as well as antibodies that recognized differential phosphorylated states of rat NF-H and NF-M. All antibodies tested showed enhanced immunostaining of enlarged axons and of large spheroids in the spinal cord and dorsal root ganglia of experimental animals. Biochemical analyses of IDPN-treated animals revealed enrichment of NF-H, NF-M, and NF-L in homogenates of dorsal root ganglia and of proximal motor and sensory nerve roots as well as depletion of the three subunits in distal nerve roots and in sciatic nerves. Immunoblots revealed a uniform enrichment of NF-H, NF-M, and NF-L in NF aggregates as well as the same admixture of phosphorylated and dephosphorylated epitopes of NF-H and NF-M in experimental and in control tissues. The global increase of immunoreactivity in axonal swellings to antibodies that react with phosphorylated, nonphosphorylated,and phosphorylation-independent NF epitopes suggests that IDPN induces an accumulation of NFs in proximal axons without necessarily altering the state of NF phosphorylation.     

Non-phosphorylated and phosphorylated neurofilaments in hypothyroid rat cerebellum

Purkinje cell baskets in hypothyroid rat cerebellum were studied with antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes. Compared to normal rats, Purkinje cell baskets were fewer in number and less developed in hypothyroid rat cerebellum. However, no differences were observed as to their immunoreactivity with monoclonal antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes.     

Effect of the substrate on neurofilament phosphorylation in mixed cultures of rat embryo spinal cord and dorsal root ganglia.

The effect of the substrate on neurofilament phosphorylation was studied in primary cultures of spinal cord and dorsal root ganglia dissociated from 15-day-old rat embryos. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites. (Primaria dishes have a modified plastic surface with a net positive charge). On both polylysine and Primaria substrates, spinal cord neurons were stained with neurofilament monoclonal antibodies reacting with phosphorylated epitopes appearing early in rat embryo development, i.e. soon after neurofilament expression. Conversely, immunoreactivity with antibodies recognizing late phosphorylation events was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, was only observed on Primaria substrates. As reported by many investigators, fibronectin and laminin were excellent substrates for dorsal root ganglia neurons in culture. However, on both laminin and fibronectin substrates immunoreactivity with antibodies recognizing late phosphorylation events, only occurred after several days in culture, at a time when non-neuronal cells (mainly astrocytes) had formed a confluent monolayer.     

Inhibition of proteolysis enhances aluminum-induced perikaryal neurofilament accumulation but does not enhance tau accumulation

As observed for neuronsin situ, phosphorylated neurofilament (NF) epitopes are normally segregated within the axonal cytoskeleton of NB2a/d1 cells. However, accumulations of phosphorylated NFs develop in NB2a/d1 perikarya following, exposure to aluminum salts and following inhibition of proteolysis. In the present study, we observed that perikarya of cells exposed to both aluminum and the protease inhibitor C1 (also known as “AllNal”) were more intensely labeled by monoclonal antibodies directed against both nonphosphorylated and phosphorylated epitopes than were cells treated with either aluminum or protease inhibitor alone. Since these monoclonal antibodies crossreact with tau, we also immunostained cells treated under these conditions with monoclonal antibodies directed against phosphate-insensitive (5E2) and phosphorylated (PHF-1) epitopes of tau. Aluminum treatment, but not C1 treatment, induced accumulation of total tau isoforms as judged by an increase in 5E2 immunoreactivity Neither treatment, either separately or in combination, induced an increase in PHF-1 immunoreactivity. These findings suggest that alterations in immunoreactivity with SMI antibodies reflected increases in NF epitopes. This was confirmed by immunoblot analyses. Since proteolysis is apparently instrumental in maintaining the normal distribution patterns of phosphorylated NF epitopes, these findings implicate deficiencies in proteolytic mechanisms in the development of neurofibrillary pathology, and underscore the possibility of a multiple etiology in human neuropathological conditions.     

Novel axonal distribution of neurofilament‐H phosphorylated at the glycogen synthase kinase 3β‐phosphorylation site in its E‐segment

The Ser493 residue in the E‐segment of the rat neurofilament heavy chain (NF‐H) is phosphorylated by glycogen synthase kinase 3β (GSK3β) in vitro and in spinal cord. We examined Ser493 phosphorylation by analyzing developmental changes and cellular distribution of phospho‐Ser493 using phosphorylation‐site‐specific antibodies. This residue was phosphorylated in NF‐H prepared from human, rat, and mouse spinal cord, all species in which the amino acid sequence of NF‐H is known. Phosphorylated Ser493 appeared on postnatal day 2 in rat brain, at the same time when NF‐H is first detected. It gradually increased together with the increase in total NF‐H during brain development. Phospho‐Ser493 was detected on the phosphorylated form of NF‐H at multiple Lys‐Ser‐Pro (KSP) repeats, which are distributed mainly in axons. In rat ventral horn, phosphorylated Ser493 was localized in axons but not in cell bodies or dendrites. However, the distributions of phosphorylated Ser493 and KSP phosphorylation in axons were not identical. Ser493 was continuously phosphorylated at nodes of Ranvier, whereas the KSP sites were dephosphorylated. Ser493 was also phosphorylated in unmyelinated regions of optic nerve axons. A biochemical difference in phosphorylation between Ser493 and KSP repeats was also found; the subtle phosphorylation at Ser493 was detected in NF‐H unphosphorylated at the KSP repeats by immunoblotting cerebral cortex extracts. These results indicate that Ser493 in the NF‐H E‐segment is a novel site that is phosphorylated in both the myelinated and the unmyelinated regions of axons. © 2009 Wiley‐Liss, Inc.     

Immunocytochemical studies of neurofilament antigens in the neurofibrillary pathology induced by aluminum

Intrathecal administration of aluminum salts induces accumulation of neurofilaments in axons and perikarya of motor neurons and is associated with impaired axonal transport of neurofilament proteins. Because phosphorylation of the 200-kilodalton (kd) neurofilament protein, thought to be a major component of the sidearms, seems to be important in interactions of neurofilaments with other cytoskeletal elements, we have postulated that aluminum may produce neurofibrillary pathology by altering patterns of neurofilament phosphorylation. To test this hypothesis, antibodies against phosphorylated and non-phosphorylated neurofilament epitopes were used for immunocytochemical analysis of spinal cord sections from aluminum-injected rabbits. In control animals, phosphorylated 200-kd neurofilament proteins were not demonstrable in perikarya of motor neurons. In experimental rabbits, perikarya and proximal axons of affected motor neurons showed striking accumulations of immunoreactivity of one phosphorylated epitope. The presence of phosphorylated 200-kd neurofilament proteins in these regions may have important consequences for the organization of the cytoskeleton and for the transport of neurofilaments. A similar, but not identical, pattern of accumulation of phosphorylated neurofilament immunoreactivity has recently been observed in neurofibrillary tangles in Alzheimer's disease.