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.     

Mice with disrupted midsized and heavy neurofilament genes lack axonal neurofilaments but have unaltered numbers of axonal microtubules.

Mammalian neurofilaments are assembled from the light (NF-L), midsized (NF-M), and heavy (NF-H) neurofilament proteins. While NF-M and NF-H cannot self-assemble into homopolymers, the data concerning NF-L has been more contradictory. In vitro bovine, porcine, and murine NF-L can homopolymerize in the absence of other subunits. However, in vivo studies suggest that neither rat nor mouse NF-L can form filaments when transfected alone into cells lacking endogenous intermediate filaments. By contrast, human NF-L forms homopolymers in similar cell lines. Recently we generated mice with null mutations in the NF-M and NF-H genes. To determine if mouse NF-L can homopolymerize in mouse axons, NF-M and NF-H null mutants were bred to create a line of double mutant animals. Here we show that axons in NF-M/H double mutant animals are largely devoid of 10-nm filaments. Instead, the axoplasm is transformed to a microtubule-based cytoskeleton-although the lack of any increase in tubulin levels per unit length of nerve or of increases in microtubule numbers relative to myelin sheath thickness argues that microtubules are not increased in response to the loss of neurofilaments. Thus in vivo rodent neurofilaments are obligate heteropolymers requiring NF-L plus either NF-M or NF-H to form a filamentous network.     

mRNA levels of all three neurofilament proteins decline following nerve transection

The control of neurofilament (NF) protein gene expression was studied by determining and comparing the levels of mRNA to the heavy (NF-H), mid-sized (NF-M) and light (NF-L) NF protein subunits in rat dorsal root ganglia (DRG) following sciatic nerve transection. mRNA to NF-H (4.5 kb), to NF-M (3.4 kb) and to NF-L (2.5 and 4.0 kb) were identified in Northern blots and quantitated in dot blot analyses, using specific cDNA probes for each NF protein. Following transection and continuing for at least 28 days. The early and co-terminal fall in mRNAs suggests that the 3 NF genes are regulated by common factor(s) and that the function of these factor(s) is influenced by the state of axonal continuity with the target organ.     

Individual neurofilament subunits reassembled in vitro exhibit unique biochemical, morphological and immunological properties

Purified bovine neurofilament (NF) subunit proteins were reassembled in vitro to form either homopolymeric or heteropolymeric intermediate-sized filaments using single or paired combinations of NF triplet proteins. Using conditions established for the reassembly of bovine NF triplet proteins, we demonstrated that the low Mr NF subunit (NF-L) alone and in combination with the middle Mr NF subunit (NF-M) reassembled very efficiently, i.e. greater than 95% of these proteins formed filaments within 90 min from the start of reassembly. In contra-distinction, the high Mr NF subunit (NF-H) alone and in combination with NF-M or NF-L underwent reassembly to a lesser extent, i.e. 62-88% of these proteins reassembled within 90 min. Immunolabeling of the reassembled NF polymers revealed striking differences in the organization of rod domain determinants. Specifically, antibodies specific for epitopes in the rod domains of NF-H, NF-M and NF-L failed to bind heteropolymeric filaments but recognized rod domains in the homopolymers. In contrast, antibodies specific to head and tail domains of all NF proteins labeled the reassembled hetero- and homopolymeric NFs. Double-labeling of heteropolymers demonstrated that pairs of different NF subunits coassembled into intermediate-sized filaments. Our results also showed that only copolymeric filaments of NF-L and NF-M, but not NF-L/NF-H and NF-M/NF-H were able to form long and stable 10-nm wide filaments. These observations provide new insights into the requirements for stable filament formation from NF subunits. In particular, they support the notion that only NF-L/NF-M, but not NF-L/NF-H or NF-M/NF-H might assemble into a stable filamentous network in vivo.     

Two-stage expression of neurofilament polypeptides during rat neurogenesis with early establishment of adult phosphorylation patterns

Monoclonal antibodies (mAbs) to rat neurofilament (NF) proteins NF-L, NF-M, and NF-H were used to examine the developmental programs of NF expression in rat embryos. The ability of these mAbs to recognize differentially phosphorylated states of NF-M and NF-H (Lee et al., 1987, the preceding paper) was exploited in order to examine the temporal and spatial patterns of NF phosphorylation during early neuronal development in vivo. NF proteins were first detected on the twelfth day postfertilization (E12) using NF-L- or NF-M-specific mAbs. By E13, the coexpression of NF-L and NF-M was widespread, reflecting dramatic increases of immunoreactivity to both subunits. Partial phosphorylation, denoted P[+], of NF-M was already present in perikarya and neurites of E12 neurons. Extensively phosphorylated, or P[+++], isoforms of NF-M appeared in E13 axons, thereby establishing a proximodistal gradient of NF phosphorylation during the earliest phase of NF expression. Immunoblots of tissue homogenates revealed that most NF-M of E13 embryos exists in a partially phosphorylated, or P[+], isoform. Unequivocal staining for NF-H first appeared at E15, a time at which NF-L and NF-M had already attained their adult patterns of immunocytochemical staining. Levels of NF-H were extremely low at E15 but could be detected in all of its differentially phosphorylated states, i.e., nonphosphorylated P[-], partly P[+], and highly P[+++] phosphorylated isoforms. P[+++] isoforms of NF-H were restricted to the distal portions of E15 axons, although staining of more proximal axons, like those in adult, was noted by E17. Immunoblots of E17 embryos revealed most NF-H as P[-] and P[+] isoforms. Quantities of immunoreactive NF-H increased very slowly and remained well below those of NF-M and NF-L for several weeks beyond birth. These results show that sequential forms of NFs are expressed by developing and maturing neurons throughout the nervous system. An "immature" form of NFs, composed of NF-M and NF-L, appears to function in establishing the neuronal phenotype and in initiating and maintaining neurite outgrowth. Addition of NF-H confers a "mature" state to the NF. This delayed expression of NF-H is a slow and graduated process that coincides in time with the stabilization of neuronal circuitries and may be important in modulating axonal events, such as the slowing of cytoskeletal transport and the growth of axonal caliber.     

Participation of neurofilament proteins in axonal dark degeneration of rat's optic nerves

Neurofilaments (NF) are neuronal intermediate filaments formed by three different subunits: high (NF-H), medium (NF-M) and light (NF-L). They are responsible for the determination and maintenance of axon caliber. Accumulation of NF or their immunoreactive products are components of several neurodegenerative disease lesions, such as neurofibrillary tangles, Lewy bodies and the spheroids of amyotrophic lateral sclerosis. Also, cytoskeletal breakdown is one of the first ultrastructural changes occurring after nerve crush or section. In the present study, Wistar rats were subjected to bilateral enucleation to induce Wallerian degeneration of optic nerve fibers and perfused 24 h, 48 h and 1 week later. Optic nerve segments were processed for electron microscopy (EM), light microscopy immunofluorescence (LM) and immunoelectronmicroscopy (IEM) for NF subunit detection. LM for NF of control nerves showed a slightly different pattern and intensity for each subunit, with more intense staining of NF-M and NF-H and less intense staining of NF-L. This reaction did not change considerably at 48 h, but was severely reduced 1 week after enucleation. Results of EM showed fibers in: (1) partial cytoskeleton degeneration or (2) watery degeneration or (3) dark degeneration. The number of dark degenerating axons was statistically higher at the latest time-interval studied. Neurofilament clumping areas and dark degenerating axons showed positive immunostaining for the three neurofilaments subunits when examined by IEM. These results suggest that dark degenerating axons develop from areas of neurofilament aggregation. We may also conclude that NF proteins participate in the process of axonal dark degeneration.     

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.     

Cloning and developmental expression of the murine neurofilament gene family

DNA clones encoding the 3 mouse neurofilament (NF) genes have been isolated by cross-hybridization with a previously described NF-L cDNA probe from the rat. Screening of a lambda gt10 cDNA library prepared from mouse brain RNA led to the cloning of an NF-L cDNA of 2.0 kb that spans the entire coding region of 541 amino acids and of an NF-M cDNA that covers 219 amino acids from the internal alpha-helical region and the carboxy-terminal domains of the protein. These cDNA clones were used as probes to screen mouse genomic libraries, and cosmid clones containing both NF-L and NF-M sequences were isolated as well as overlapping cosmids containing the NF-H gene. This strongly suggests that the 3 neurofilament genes are organised in a cluster and derived by gene duplication of a common ancestral gene. RNA blot analyses using specific DNA probes for each of the genes indicate that NF mRNAs are differentially expressed during brain development. The NF-L and NF-M mRNAs are detected early in the embryonal brain, with a progressive increase in their levels during development, while the NF-H mRNA is barely detectable at embryonal stages and accumulates later in the postnatal brain.     

Monoclonal antibodies distinguish several differentially phosphorylated states of the two largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats

A new panel of greater than 300 monoclonal antibodies (mAbs) was prepared to the high, middle, and low Mr rat neurofilament (NF) subunits (NF-H, NF-M and NF-L, respectively). NF proteins were purified both from native, i.e., phosphorylated rat NFs and from enzymatically dephosphorylated rat NFs. The resulting mAbs were used to biochemically and immunochemically distinguish and characterize distinct and differentially phosphorylated isoforms of NF subunits. By immunoblot, all mAbs specific for NF-L and some mAbs specific for NF-M detected their specific NF subunit regardless of whether or not the NFs had been treated with alkaline phosphatase, and such antibodies were termed "phosphate-independent" or P[ind] mAbs. The other mAbs were specific for NF-M, NF-H, or for both NF-M and NF-H, and they recognized epitopes in the COOH termini of these subunits. Significantly, the latter mAbs could discriminate different isoforms of NF-M and NF-H, depending on the phosphorylation state of each variant. Such mAbs were assigned to one of 4 distinct categories on the basis of their performance in immunoblots of progressively dephosphorylated rat NF samples and by immunohistochemistry of various adult rat nervous tissues: (1) P[-] mAbs preferentially stained neuronal perikarya and dendrites, and they recognized only extensively dephosphorylated (and nonphosphorylated) NF-H; (2) P[+] mAbs stained axons more strongly than perikarya, and primarily blotted phosphorylated, but not nonphosphorylated, forms of NF-H and NF-M; (3) P[++] mAbs stained axons almost to the exclusion of perikarya, and in blots recognized only the extensively phosphorylated forms of NF-H and NF-M (i.e., subunits subjected to limited enzymatic dephosphorylation); (4) P[ ] mAbs also predominantly stained axons, but the briefest alkaline phosphatase treatment abolished the NF-M and NF-H immunobands produced by these mAbs. Two-dimensional gel analysis and immunoblotting of total proteins from adult rat dorsal root ganglion verified mAb specificity in situ, and showed that differentially phosphorylated isoforms of NF-M and NF-H occur in vivo. This provided additional evidence that mAbs can detect all 4 phosphorylation-dependent endogenous isoelectric variants of NF-H and NF-M.(ABSTRACT TRUNCATED AT 400 WORDS)     

Early posttranslational modifications of the three neurofilament subunits in mouse retinal ganglion cells: neuronal sites and time course in relation to subunit polymerization and axonal transport

We have characterized stages in the posttranslational processing of the three neurofilament subunits, High (NF-H), Middle (NF-M), and Low (NF-L), in retinal ganglion cells in vivo during the interval between synthesis in cell bodies within the retina and appearance of these polypeptides in axons at the level of the optic nerve (optic axons). Neurofilament proteins pulse-labeled by injecting mice intravitreally with [35S]methionine or [32P]orthophosphate, were isolated from Triton-soluble and Triton-insoluble fractions of the retina or optic axons by immunoprecipitation or immunoaffinity chromatography. Within 2 h after [35S]methionine injection, the retina contained neurofilament-immunoreactive radiolabeled proteins with apparent molecular weights of 160, 139, and 70 kDa, which co-migrated with subunits of axonal neurofilaments that were dephosphorylated in vitro with alkaline phosphatase. The two larger polypeptides were not labeled with [32P]orthophosphate, indicating that they were relatively unmodified forms of NF-H and NF-M. About 75% of the subunits were Triton-insoluble by 2 h after isotope injection, and this percentage increased to 98% by 6 h. Labeled neurofilament polypeptides appeared in optic axons as early as 2 h after injection. These subunits exhibited apparent molecular weights of 160, 139, and 70 kDa and were Triton-insoluble. The time of appearance of fully modified polypeptide forms differed for each subunit (2 h for NF-L, 6-18 h for NF-M, 18-24 h for NF-H) and was preceded by the transient appearance of intermediate forms. The modified radiolabeled subunits in optic axons 3 days after synthesis were heavily labeled with [32P]orthophosphate and exhibited the same apparent molecular weights as subunits of axonal neurofilaments (70 kDa, 145 and 140 kDa, and 195-210 kDa, respectively). Whole mounts of retina immunostained with monoclonal antibodies against NF-H in different states of phosphorylation demonstrated a transition from non-phosphorylated neurofilaments to predominantly phosphorylated ones within a region of the axon between 200 and 1000 microns downstream from the cell body. These experiments demonstrate that the addition of most phosphate groups to NF-M and NF-H takes place within a proximal region of the axon. The rapid appearance of modified forms of NF-L after synthesis may imply that processing of this subunit occurs at least partly in the cell body. The presence of a substantial pool of Triton-insoluble, unmodified subunits early after synthesis indicates that the heaviest incorporation of phosphate occurs after neurofilament proteins are polymerized.(ABSTRACT TRUNCATED AT 400 WORDS)     

Some neural intermediate filaments contain both peripherin and the neurofilament proteins.

Mammalian neurons and neuron-like cultured cells express the neural intermediate filament (IF) proteins neurofilament (NF)-L, NF-M, NF-H, and peripherin. To determine whether these proteins are found within the same 10-nm filament, light and electron microscope immunocytochemistry using peripherin and NF-specific antibodies was performed on PC12 cells, nervous tissue, and isolated neural filaments from the cauda equina. Double-label immunofluorescence showed that peripherin and NF-L, -M, and -H were found in identical filamentous patterns in interphase and mitotic PC12 cells. Furthermore, expression of mutant peripherin in PC12 cells disrupted not only the peripherin network but also NF-containing filaments. Immunoelectron microscopy of PC12 cell cytoskeletons showed that peripherin and NF subunit proteins were found in the same filament. In situ, in the sciatic nerve, peripherin/NF-L or peripherin/NF-M/-H double-label immunofluorescence illustrates at least three types of nerve fibers: those containing NF only, those labeled predominantly for peripherin, and fibers in which peripherin and NF subunits were colocalized. Immunoelectron microscopy of filaments isolated from nerve roots comprising the sciatic nerve also showed the same three labeling patterns seen by light microscopy. Some neural IF appear to contain predominantly NF proteins or peripherin, but in others, both proteins are found within the same IF.     

Neurofilaments of aged rats: the strengthened interneurofilament interaction and the reduced amount of NF-M.

Amyotrophic lateral sclerosis is an age-related neurological disease, characterized by neurofilament (NF) accumulation in primary axons followed by degeneration of motor neurons. To elucidate age-related factors that might lead to pathological NF accumulation, NFs were compared between young and aged rats. Electron microscopic examination of sciatic nerve axons revealed that NFs were more than twice as densely packed in aged rat axons (542 +/- 180 NFs/mm2) as in young adult rat axons (211 +/- 73 NFs/mm2). The NFs isolated from aged rats also appeared to be more aggregated than those from young rats. Phosphorylation at the head or tail domains was studied as a possible candidate affecting NF organization. Western blotting with phosphorylation-dependent antibodies showed higher phosphorylation of NF-H in the tail domains of aged rat spinal cord NFs, but dephosphorylation did not diminish the differences in aggregation between aged and young rat NFs. On the other hand, when NFs were phosphorylated by A-kinase on their head domains, the extent of phosphorylation in NF-M of aged rat NFs was only one-third of young rat NFs. We found that aged rat NFs contained only 60% of the NF-M of young rat NFs in molar ratio compared to NF-L. These results raise a possibility that the decreased amount of NF-M induces the aggregates of isolated NFs and the higher packing density of NF in aged rat axons.     

Electrophysiological properties of axons in mice lacking neurofilament subunit genes: disparity between conduction velocity and axon diameter in absence of NF-H

Neurofilament proteins (NFs) are made by co-polymerization of three intermediate filament proteins, NF-L, NF-M and NF-H and constitute the most abundant cytoskeletal element in large myelinated axons. NFs have a well-established role as intrinsic determinants of axon caliber with all the functional implications, but the role of each individual NF subunit is much less clear. The aim of our study was to examine functional properties of large myelinated axons with altered morphology from mice bearing a targeted disruption of each NF genes (NF-L -/-, NF-M-/- and NF-H -/- mice). Membrane properties, action potentials and single axon refractory period were measured in isolated sciatic nerves in vitro, using intra-axonal microelectrode recording in conjunction with current-clamp technique. Some results were obtained from whole nerves by sucrose-gap recording. The NF-knockout mice showed several deficits in physiological properties of low-threshold fibers. In keeping with smaller axon diameter, the conduction velocity was significantly decreased in NF-L -/- and NF-M -/- transgenic animals (control, 39.9+/-1.8 m/s, NF-M -/-; 23.5+/-1. 4 m/s, and NF-L-/-; 12.0+/-0.7 m/s, mean+/-S.E.M.; intra-axonal recording; similar ratios obtained by sucrose-gap recording; 22-26 degrees C). However, in spite of their preserved caliber, large myelinated axons in NF-H -/- mice also showed a significant decrease in conduction velocity (22.8+/-1.0 m/s, mean+/-S.E.M.). Although action potential amplitudes, duration and shape did not differ between control axons and transgenic animals, the refractory period was prolonged in NF-H -/- and NF-M -/- animals. Intracellular injections of 200 ms depolarizing and hyperpolarizing currents revealed outward and inward rectification in all animal groups. In comparison to control animals, NF-H -/- mice expressed a significant decrease in outward rectification. Potassium channel blockers (4AP and TEA) and cesium ions were able to block outward and inward rectification in all myelinated axons in qualitatively the same manner. These results suggest that NF-H may have a specific role in modulating ion channel functions in large myelinated fibers.     

Differential expression and modification of neurofilament triplet proteins during cat cerebellar development

Neurofilament (NF) proteins consist of three subunits of different molecular weights defined as NF-H, NF-M, and NF-L. They are typical structures of the neuronal cytoskeleton. Their immunocytochemical distribution during postnatal development of cat cerebellum was studied with several monoclonal and polyclonal antibodies against phosphorylated or unmodified sites. Expression and distribution of the triplet neurofilament proteins changed with maturation. Afferent mossy and climbing fibers in the medullary layer contained NF-M and NF-L already at birth, whereas NF-H appeared later. Within the first three postnatal weeks, all three subunits appeared in mossy and climbing fibers in the internal granular and molecular layers and in the axons of Purkinje cells. Axons of local circuit neurons such as basket cells expressed these proteins at the end of the first month, whereas parallel fibers expressed them last, at the beginning of the third postnatal month. Differential localization was especially observed for NF-H. Depending on phosphorylation, NF-H proteins were found in different axon types in climbing, mossy, and basket fibers or additionally in parallel fibers. A nonphosphorylated NF-H subunit was exclusively located in some Purkinje cells at early developmental stages and in some smaller interneurons later. A novel finding is the presence of a phosphorylation site in the NF-H subunit that is localized in dendrites of Purkinje cells but not in axons. Expression and phosphorylation of the NF-H subunit, especially, is cell-type specific and possibly involved in the adult-type stabilization of the axonal and dendritic cytoskeleton. © 1996 Wiley-Liss, Inc.     

Nerve regeneration along collagen filament and the presence of distal nerve stump

This article describes the regeneration of severed peripheral nerve axons along collagen filaments in the absence of the distal nerve stump. 22-mm long nerve guides made of collagen filaments were sutured to the proximal ends of severed rat sciatic nerves. The distal ends of the guides were sutured to the distal stumps of the nerves in a group and not sutured in the other. Nerve autografts and collagen tubes were used as controls. At 8 weeks postoperatively, the mean number and the mean diameter of myelinated axons were 5491 +/- 617 (mean +/- SD) and 2.3 +/- 1.3 microns at the distal ends of the collagen filaments nerve guides those the distal ends were sutured to the distal stumps of the nerves, while in the nerve autografts these were 4837 +/- 604 and 3.3 +/- 1.4 microns. These were 1992 +/- 770 and 2.7 +/- 1.2 microns at the distal ends of the collagen-filaments guides those the distal ends were not sutured to the distal stumps of the nerves, while in the nerve autografts these were 3041 +/- 847 and 2.3 +/- 1.1 microns. No axon was found at the distal ends of the collagen tubes. The results suggested that the contact guidance and the chemotaxis guided regenerating axons along the collagen filaments.     

Constitutive expression of the mature array of neurofilament proteins by a CNS neuronal cell line.

Neurofilament protein expression was examined immunochemically in a neuronal cell line derived from postnatal day 21 septal tissue. The SN48.1p cell line was found to constitutively synthesize an array of neurofilament proteins typical of a mature neuron. All three neurofilament subunits (NF-L, NF-M, and NF-H) as well as differentially phosphorylated isoforms (P-, P+, P++, and P ) of NF-M and NF-H were identified by immunoblot analysis. Immunofluorescence studies revealed that the neurofilament proteins were components of discrete, filamentous structures. Abnormal intracellular aggregations of neurofilament proteins were never observed. Some SN48.1p cells apportioned specific isoforms into selected intracellular regions based on the molecular weight and phosphorylation level of the protein. NF-L was preferentially localized to perikarya and proximal neurites; NF-M[P++] and NF-H[P ] were distributed to distal aspects of neurites. The expression of these differentiated features of neurofilament proteins and, presumably, the synthesis of the kinases and phosphatases required for normal neurofilament metabolism occurred in the absence of growth factors, differentiating agents, and specialized culture substrates. In addition, the non-neuronal intermediate filaments glial fibrillary acidic protein and epithelial cytokeratin proteins were absent. These data demonstrate that SN48.1p cells exhibit a neurofilament phenotype characteristic of mature neurons and provide a unique model to examine the expression and function of neurofilaments in differentiated neuronal cells.     

Simultaneous up-regulation of neurofilament proteins during the postnatal development of the rat nervous system

The expression of neurofilament (NF) proteins was examined during postnatal development of the rat nervous system in order to elucidate the nature of NF expression during the period of transition from the embryonic (immature) to the adult (mature) stages of NF expression. mRNAs to the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF proteins were compared by Northern blots and by in situ hybridizations, in NF-rich (i.e., DRG, spinal cord and brainstem) and in NF-poor (i.e., cerebellum and cerebral cortex) regions of the developing rat nervous system. NF proteins were analyzed by gel electrophoresis and by immunoblots. In each tissue, the expression of NF-H was delayed compared to that of NF-L and NF-M, as previously reported. The present study now shows that the delayed expression of NF-H is accompanied by parallel up-regulations in the expressions of NF-L and NF-M, both at the levels of mRNA and protein. Similar rates of increase of all three NF mRNAs occur between postnatal days 5 (P5) and 24 (P24) in rat spinal cord and DRG. Furthermore, the postnatal up-regulation of NF expression is characterized by a progressive accumulation of all three NF proteins in the tissues. The findings indicate that the adult pattern of NF expression (with high levels of expression of all three NF proteins) becomes established during the postnatal period of development, raising questions as to the nature of factors that coregulate the expression of NF subunits.     

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.     

Thyroid hormones stimulate expression and modification of cytoskeletal protein during rat sciatic nerve regeneration

Peripheral neurons can regenerate after axotomy; in this process, the role of cytoskeletal proteins is important because they contribute to formation and reorganization, growth, transport, stability and plasticity of axons. In the present study, we examined the effects of thyroid hormones (T3) on the expression of major cytoskeletal proteins during sciatic nerve regeneration. At various times after sciatic nerve transection and T3 local administration, segments of operated nerves from T3-treated rats and control rats were examined by Western blotting for the presence of neurofilament, tubulin and vimentin. Our results revealed that, during the first week after surgery, T3 treatment did not significantly alter the level of NF subunits and tubulin in the different segments of operated nerves compared to control nerves. Two or 4 weeks after operation, the concentration of NF-H and NF-M isoforms was clearly increased by T3 treatment. Moreover, under T3-treatment, NF proteins appeared more rapidly in the distal segment of operated nerves. Likewise, the levels of betaIII, and of acetylated and tyrosinated tubulin isotypes, were also up-regulated by T3-treatment during regeneration. However, only the tyrosinated tubulin form appeared earlier in the distal nerve segments. At this stage of regeneration, T3 had no effect on the level of vimentin expression. In conclusion, thyroid hormone improves and accelerates peripheral nerve regeneration and exerts a positive effect on cytoskeletal protein expression and transport involved in axonal regeneration. These results help us to understand partially the mechanism by which thyroid hormones enhance peripheral nerve regeneration. The stimulating effect of T3 on peripheral nerve regeneration may have considerable therapeutic potential.