Immunohistochemical differences between neurofilaments in perikarya, dendrites and axons : Immunofluorescence study with antisera raised to neurofilament polypeptides (200K, 150K, 70K) isolated by anion exchange chromatography

Neurofilament (NF) proteins (70K, 150K and 200K D) were isolated from 2 M urea extracts of bovine spinal cord by anion exchange chromatography. Antisera to the individual NF polypeptides were produced in rabbits and affinity-purified on Sepharose columns prepared with their own antigen. The NF antisera were completely absorbed by their own antigen at protein concentrations that did not decrease the staining when the absorption was conducted with the heterologous NF antigens. Partial absorption (decrease in immunofluorescence titer) occurred at higher concentrations of the heterologous antigens. Cross-reactivity between the polypeptides of the NF triplet could not be detected by double immunodiffusion. The antisera formed immunoprecipitin lines only when reacted with their own antigen. Conversely, cross-reactivity was demonstrated by the immune blotting procedure. Anti-70K stained all three NF polypeptides. Anti-200K and anti-150K stained both 200K and 150K but not 70K, the main reaction being with their own antigen. The antisera were rendered monospecific by adsorption of the common antigenic determinants on Sepharose columns prepared with the heterologous NF antigens. The localization of the NF proteins was studied by immunofluorescence on cryostat sections of rat brain, cerebellum, spinal cord and posterior root ganglia. All NF antisera (anti-70K, anti-150K and anti-200K) stained axons including Purkinje cell baskets with identical pattern. Spinal cord motor neurons, posterior root ganglia neurons and pyramidal neurons in the cerebral cortex stained with anti-70K and anti-200K. No staining of neuronal perikarya and dendrites was observed with anti-150K. Aluminium-induced neurofibrillary tangles in rabbit spinal cord stained with anti-70K and anti-200K. The tangles were not decorated by anti-150K. It is concluded that a marked difference exists in the concentration of 150K depending on the location, i.e., cell body or axon; or, alternatively, that 150K undergoes modification of antigenic sites within the axon so that it may not be recognized immunologically as a component of the neurofilament within perikarya and dendrites.     

Maturation of neurites in mixed cultures of spinal cord neurons and muscle cells from Xenopus laevis embryos followed with antibodies to neurofilament proteins

Dissociated cell cultures of Xeopus laevis embryonic spinal cord have proved useful for studying the differentiation of neuronal ionic channel and membrane properties and for examining the dynamics of microtubules in developing neurons. To examine their usefulness for studying neurofilaments in developing neurites, we prepared similar cultures from stage 22 embryos. Between 3 and 55 h after plating, these cultures were fixed and immunostained with antibodies directed against various epitopes of neurofilament proteins from X. Laevis. These antibodies were specific for nonphosphorylated epitopes of the two low molecular weight Xenopus neurofilament proteins (Xenopus NF-L and the Xenopus neuronal intermediate filament protein, XNIF), both phosphorylated and nonphosphorylated epitopes of the Xenopus middle molecular weight neurofilament protein (NF-M), and a nonphosphorylated epitope of the Xenopus high molecular weight neurofilament protein (NF-H). The emergence of these neurofilament proteins in culture was compared to the time course previously reported for them in Xenopus spinal cord neurons in situ. To facilitate the comparison of times in culture to developmental stages, the age of cultured neurons was converted to an equivalent Nieuwkoop and Faber normal stage using data presented here on the effect of changing temperature on developmental rates of X. laevis. With the exception of the nonphosphorylated epitope of NF-H, which is indicative of the most mature axons found in situ. the emergence of the other neurofilament protein antibody epitopes closely paralleled that previously reported for these antibodies in situ. Thus, with respect to XNIF, NF-M, and NF-L, the neurities of cultured neurons were typical of young embryonic Xenopus laevis spinal cord axons. This system should prove useful for studying both the function of these neurofilament proteins during the early stages of axonal development and the dynamics of their transport. 1994 John Wiley & Sons, Inc.     

Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits

To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we generated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large myelinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF-M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two-fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate filaments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approximately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and alpha-internexin proteins were reduced dramatically throughout the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed enhanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [35S]methionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined results demonstrate a requirement of the high-molecular-weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment.     

Presence of an alpha-melanocyte-stimulating hormone-like epitope in the 150,000 dalton neurofilament subunit from diverse regions of the central nervous system: an immunohistochemical and immunoblot study in guinea pig

Monoclonal antibodies specific for the two higher molecular weight neurofilament (NF) subunits (NF200 and NF150), and antiserum to alpha-melanocyte-stimulating hormone (alpha-MSH) were used to probe the distribution of an alpha-MSH-like epitope in NF proteins of the guinea pig central nervous system using immunoblot and immunohistochemical methods. The anti-alpha-MSH antiserum recognized the same protein band as an anti-NF150 monoclonal antibody in immunoblots of proteins extracted from guinea pig cerebellum, spinal cord, retina, optic nerve, and neurohypophysis; it also stained axons and dendrites in sections of cerebellum, retina, and optic nerve. Although all cells of the pars intermedia and some in the pars distalis exhibited immunoreactivity with this antiserum, it did not stain axons in the neurohypophysis. Our immunoblot data demonstrate an alpha-MSH-like epitope in NF150 extracted from each of the regions studied. The lack of in situ recognition of this alpha-MSH-like epitope in neurophypophyseal axons, using the same immunohistochemical methods that demonstrate this epitope in axons of the cerebellum, retina, and optic nerve, suggests that NF150 is immunochemically heterogeneous in different regions of the guinea pig central nervous system.     

Hybrid character of a large neurofilament protein (NF-M): intermediate filament type sequence followed by a long and acidic carboxy-terminal extension

The sequence of the amino-terminal 436 residues of porcine neurofilament component NF-M (apparent mol. wt. in gel electrophoresis 160 kd), one of the two high mol. wt. components of mammalian neurofilaments, reveals the typical structural organization of an intermediate filament (IF) protein of the non-epithelial type. A non-alpha-helical arginine-rich headpiece with multiple beta-turns (residues 1-98) precedes a highly alpha-helical rod domain able to form double-stranded coiled-coils (residues 99-412) and a non-alpha-helical tailpiece array starting at residue 413. All extra mass of NF-M forms, as a carboxy-terminal tailpiece extension of approximately 500 residues, an autonomous domain of unique composition. Limited sequence data in the amino-terminal region of this domain document a lysine- and particularly glutamic acid-rich array somewhat reminiscent of the much shorter tailpiece extension of NF-L (apparent mol. wt. 68 kd), the major neurofilament protein. NF-M is therefore a true intermediate filament protein co-polymerized with NF-L via presumptive coiled-coil type interactions and not a peripherally bound associated protein of a filament backbone built exclusively from NF-L. Along the structurally conserved coiled-coil domains the two neurofilament proteins show only approximately 65% sequence identity, a value similar to that seen when NF-L and NF-M are compared with mesenchymal vimentin. The highly charged and acidic tailpiece extensions of all triplet proteins particularly rich in glutamic acid seem unique to the neurofilament type of IFs. They could form extra-filamentous scaffolds suitable for interactions with other neuronal components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

Organization of mammalian neurofilament polypeptides within the neuronal cytoskeleton

Neurofilaments in the axons of mammalian spinal cord neurons are extensively cross-linked; consequently, the filaments and their cross- bridges compose a three-dimensional lattice. We have used antibody decoration in situ combined with tissue preparation by the quick- freeze, deep-etch technique to locate three neurofilament polypeptides (195, 145, and 73 Kd) within this lattice. When antibodies against each polypeptide were incubated with detergent-extracted, formaldehyde-fixed samples of rabbit spinal cord, each antibody assumed a characteristic distribution: anti-73-Kd decorated the neurofilament core uniformly, but not the cross-bridges; anti-145-Kd also decorated the core, but less uniformly; sometimes the anti-145-Kd antibodies were located over the bases of cross-bridges. In contrast, anti-195-Kd primarily decorated the cross-bridges between the neurofilaments. These observations show that the 73-Kd polypeptide is a component of the central core of neurofilaments, and that the 195-Kd polypeptide is a component of the inter-neurofilamentous cross-bridges. It is consistent with this conclusion that we found few cross-bridges between neurofilaments in the optic nerves of neonatal rabbits during a developmental period when the ratio of 195 to 73 or 145-Kd polypeptides is much lower than in adults. The ratio of 195-Kd polypeptide to the other two neurofilament polypeptides also appeared much lower in the cell bodies and dendrites than in axons of adult spinal cord neurons, when the dispositions of the three polypeptides were studied by immunofluorescence experiments. The cell bodies apparently contain neurofilaments composed primarily of 145- and 73-Kd polypeptides, because we observed antibody decoration of individual neurofilaments in the cell bodies with anti-73- and -145-Kd, but not with anti-195-Kd. We conclude that the 195-Kd polypeptide participates in a cross-linking function, and that this function is, at least in certain neurons, most prevalent in the mature axon.     

Cross-Linking of Neurofilament Proteins of Rat Spinal Cord In Vivo After Administration of 2,5-Hexanedione

The aliphatic hexacarbons n-hexane, methyl-n-butyl ketone, and 2,5-hexanedione are known to produce a peripheral neuropathy that involves an accumulation of 10-nm neurofilaments above the nodes of Ranvier in the spinal cord and peripheral nerve. In this study, rats were treated with 0.5% 2,5-hexanedione in drinking water for 180 days, and their spinal cord neurofilaments were isolated after development of the neuropathy. Visualization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a significant reduction in content of the neurofilament triplet proteins in treated animals and the presence of bands migrating at 138K and 260K that were not present in control animals. Analysis of the lanes using immunoblotting procedures and anti-70K, anti-160K, and anti-210K neurofilament antibodies revealed many cross-linked peptides. The 138K band cross-reacted with the anti-160K neurofilament antibody. This suggests that the 138K band is an intramolecular cross-link of the 160K neurofilament subunit. In addition to this peptide, there were numerous high-molecular-weight peptides immunoreactive with all three neurofilament protein antibodies. In addition to cross-linking, there was also a diminished amount of immunoreactive breakdown product of all three neurofilament proteins. This report demonstrates direct evidence of 2,5-hexanedione-induced cross-linking of neurofilament proteins in vivo, which maybe responsible for the accumulation of neurofilament proteins pathognomic of this neuropathy.     

In vivo phosphorylation of distinct domains of the 70-kilodalton neurofilament subunit involves different protein kinases

A combination of in vivo and in vitro approaches were used to characterize phosphorylation sites on the 70,000-kilodalton (kDa) subunit of neurofilaments (NF-L) and to identify the protein kinases that are likely to mediate these modifications in vivo. Neurofilament proteins in a single class of neurons, the retinal ganglion cells, were pulse-labeled in vivo by injecting mice intravitreously with [32P]orthophosphate. Radiolabeled neurofilaments were isolated after they had advanced along optic axons, and the individual subunits were separated on sodium dodecyl sulfate-polyacrylamide gels. Two-dimensional alpha-chymotryptic phosphopeptide map analysis of NF-L revealed three phosphorylation sites: an intensely labeled peptide (L-1) and two less intensely labeled peptides (L-2 and L-3). The alpha-chymotryptic peptide L-1 was identified as the 11-kDa segment containing the C terminus of NF-L. The ability of these peptides to serve as substrates for specific protein kinases were examined by incubating neurofilament preparations with [gamma-32P]ATP in the presence of purified cAMP-dependent protein kinase or appropriate activators and/or inhibitors of endogenous cytoskeleton-associated protein kinases. The heparin-sensitive, calcium- and cyclic nucleotide-independent kinase associated with the cytoskeleton selectively phosphorylated L-1 and L-3 but had little, if any, activity toward L-2. When this kinase was inhibited with heparin, cAMP addition to the neurofilament preparation stimulated the phosphorylation of L-2, and addition of the purified catalytic subunit of cAMP-dependent protein kinase induced intense labeling of L-2. At higher labeling efficiencies, the exogenous kinase also phosphorylated L-3 and several sites at which labeling was not detected in vivo; however, L-1 was not a substrate. Calcium and calmodulin added to neurofilament preparations in the presence of heparin modestly stimulated the phosphorylation of L-1 and L-3, but not L-2, and the stimulation was reversed by trifluoperazine. The selective phosphorylation of different polypeptide domains on NF-L by second messenger-dependent and -independent kinases suggests multiple functions for phosphate groups on this protein.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.     

Changes in the amounts of cytoskeletal proteins within the perikarya and axons of regenerating frog motoneurons

Changes in the amounts of tubulin, actin, and neurofilament polypeptides were found in regenerating motoneurons of grass frogs during the period of axonal elongation. Ventral roots 9 and 10 were transected unilaterally about 7 mm from the spinal cord. 35 d later, [3H]colchicine binding had decreased in the proximal stumps to approximately one-half of contralateral control values, well before the regenerating motor axons had reinnervated skeletal muscles of the hind limb. [3H]colchicine binding did not change significantly in the operated halves of the 9th and 10th spinal cord segments over a 75-d period. The relative amounts of actin, tubulin, and neurofilament polypeptides in the operated ventral roots were measured by quantitative densitometry of stained two-dimensional electrophoretic gels. Alpha-tubulin, beta-tubulin, and the 68,000 molecular weight subunit of neurofilaments (NF68) decreased within the transected ventral roots to 78%, 57%, and less than 15% of control values, respectively. The amount of actin increased to 132% of control values within the operated ventral roots, although this change was not statistically significant. Opposite changes were found within motoneuronal cell bodies isolated from the spinal cord. The relative amounts of alpha-tubulin, beta-tubulin and NF68 within axotomized perikarya increased, respectively, to 191%, 146%, and 144% of that in control perikarya isolated from the contralateral side of the spinal cord. Thus, the changes in NF68 and tubulin did not occur uniformly throughout the injured cells. The possible structural and functional consequences of these changes are discussed.     

Expression of low-molecular-weight neurofilament (NF-L) mRNA during postnatal development of the mouse brain

A regional Northern blot analysis demonstrated that the highest levels of NF-L mRNA in the adult mouse brain are present in brain stem followed by mid-brain, with lower levels found in neocortex, cerebellum, and hippocampus. The study was extended to the cellular level over the time course of postnatal development using in situ hybridization. This developmental analysis revealed that the expression of NF-L mRNA closely follows the differentiation pattern of many large neurons during postnatal neurogenesis. Neurons which differentiate early such as Purkinje, mitral, pyramidal, and large neurons of brain stem and thalamic nuclei, expressed high levels of NF-L mRNA at postnatal day 1. Early expression of NF-L mRNA may be required for the maintenance of the extensive neurofilament protein networks that are detected within the axons of larger neurons. Smaller neurons which differentiate later, such as dentate gyrus granule cells, small pyramidal and granule cells of the neocortex, and granule cells of the cerebellum, exhibit a delayed expression of NF-L mRNA.To whom to address reprint requests.     

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.     

Selective Axonal Argentaffin Staining in Rat Central Nervous System after Protein Mercuration

The mercury-silver (Hg-Ag) argentaffin technique, known to stain specifically proteins in the lateral components of triads/diads in striated muscle cells, was applied to the central nervous system of adult rats. Following fixation in glutaraldehyde, axons in white and gray matter were selectively stained, but not perikarya or their proximal axon and dendrites. Neural tissues were postfixed 24 hr in 5% (w/v) mercuric acetate in 2% (v/v) acetic acid in distilled water, stained for 12-24 hr in darkness at 37-43 C with ammoniacal silver nitrate solution, freshly prepared by adding concentrated ammonia to 60% (w/v) silver nitrate solution until a small amount of silver oxide precipitate remained undissolved. Samples were then washed with freshly prepared 5% (w/v) sodium sulfite and distilled water. All steps were carried out using dark-colored glass flasks. Samples were dehydrated with ethanol and embedded in Paraplast or Poly Bed. Electron microscopy showed the silver-reducing protein inside the axons. Methylation abolished Hg-Ag axonal reactivity indicating that carboxyl groups were necessary for silver staining. Proteins with solubility properties characteristic of neurofilament proteins were involved in Hg-Ag staining. In the cerebellum the plexus of parallel fibers in the molecular layer were not stained, while basket cell axonal processes reacted intensely. The method appears to distinguish neuronal protein variants related to cytotypic differences in cytoskeletal neurofilaments.     

Disproportional Expression of Proteolipid Protein and DM-20 in the X-Linked, Dysmyelinating Shaking Pup Mutant

The shaking pup is an X-linked canine mutant with a severe hypomyelination of the CNS. Proteolipid protein (PLP) and the related DM-20 protein were examined in this mutant by densitometric scanning of Western blots stained with PLP antiserum. In the spinal cord of 4-week-old mutants, PLP was reduced to less than 1% of the control level, which is a greater deficiency than was found for other myelin proteins. On Western blots of control spinal cord, PLP stained much more intensely than DM-20. However, on Western blots of the mutant spinal cord, a component with the electrophoretic mobility of DM-20 stained slightly more intensely with PLP antiserum than PLP itself. This component was shown to be DM-20 by its lack of reactivity with an antiserum raised to a synthetic peptide corresponding to part of the PLP sequence that is missing in DM-20. Thus PLP and DM-20 are expressed in approximately equal and greatly reduced amounts in the mutant spinal cord. Although PLP or DM-20 could not be detected in brain from the 4-week-old mutant, similar disproportional expression of these two proteins was demonstrated in both spinal cord and brain from a 10-week-old mutant pup. Immunostaining of tissue sections showed that the small amounts of PLP and/or DM-20 synthesized in the mutant are present in the thin myelin sheaths. The results suggest that the shaking pup could have a primary defect in the PLP gene leading to a severe deficiency of PLP and DM-20 as well as disproportional expression of these two proteins.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Abstract. A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.     

Neuropathological Abnormalities in Transgenic Mice Harbouring a Phosphorylation Mutant Neurofilament Transgene

Abstract: Ser⁵⁵ within the head domain of neurofilament light chain (NF-L) is a target for phosphorylation by protein kinase A. To understand further the physiological role(s) of NF-L Ser⁵⁵ phosphorylation, we generated transgenic mice with a mutant NF-L transgene in which Ser⁵⁵ was mutated to Asp so as to mimic permanent phosphorylation. Two lines of NF-L(Asp) mice were created and these animals express the transgene in many neurones of the central and peripheral nervous systems. Both transgenic lines display identical, early onset, and robust pathological changes in the brain. These involve the formation of NF-L(Asp)-containing perikaryal neurofilament inclusion bodies and the development of swollen Purkinje cell axons. Development of these pathologies was rapid and fully established in mice as young as 4 weeks of age. The two transgenic lines show no elevation of NF-L, neurofilament middle chain (NF-M), or neurofilament heavy chain (NF-H), and transgenic NF-L(Asp) represents only a minor proportion of total NF-L protein. Because other published transgenic lines expressing higher levels of wild-type NF-L do not exhibit phenotypic changes that in any way resemble those in the NF-L(Asp) mice and because the two different NF-L(Asp) transgenic lines display identical neuropathological changes, it is likely that the pathological alterations observed in the NF-L(Asp) mice are the result of properties of the mutant NF-L. These results support the notion that phosphorylation of Ser⁵⁵ is a mechanism for regulating neurofilament organisation in vivo.     

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

A panel of 10 mouse monoclonal antibodies specific for glial fibrillary acidic protein (GFA) has been isolated using porcine GFA as antigen. Although all antibodies recognize GFA purified from porcine spinal cord in the western blot technique, they can be subdivided into at least three groups on the basis of their reactivity against defined fragments of the molecule. Immunofluorescence staining patterns with the monoclonal antibodies performed on tissues and cell lines resemble those reported with conventional polyclonal antibodies directed against GFA. In particular astrocytes and Bergmann glia are strongly stained. In addition mouse monoclonal antibodies specific for either the 200 kd, or the 160 kd, or the 68 kd neurofilament triplet protein have been isolated and characterized. These antibodies are specific for neuronal cells and support conclusions made with similar antigen affinity-purified polyclonal antibodies. The combined set of monoclonal antibodies seems a valuable tool to characterize the different cell types of the nervous system.