Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.     

Difference in phosphorylation of neurofilament proteins in motor neurons and spinal ganglion cells

The composition of the neurofilament proteins (NFPs) in neuronal perikarya was examined by two-dimensional (2-D) gel electrophoresis of isolated perikarya of bovine spinal motor neurons. The extent of phosphorylation of the high molecular weight subunit of NFP (NFP-H) was compared between motor and sensory neuronal perikarya in spinal cord and spinal ganglion by immunocytochemistry with monoclonal antibodies (MAbs) to NFP. Of the 23 MAbs used in this study, one MAb (82E10) was specific to the highly phosphorylated component of NFP-H examined by 2-D immunoblot whereas another MAb (3A8) was specific to NFP-H irrespective of its level of phosphorylation. Immunocytochemically, 82E10 did not stain the perikarya of bovine and rabbit spinal motor neurons but 3A8 stained the perikarya in both animal species. These findings are consistent with 2-D immunoblot of neuronal perikarya of bovine motor neurons isolated in bulk. As for the spinal ganglia, 82E10 stained many, but not all, perikarya of sensory neurons of both animal species. These results indicate that the extent of phosphorylation of NFP-H in the perikarya of most spinal ganglion cells is higher than that of motor neurons. These findings suggest that the rate of phosphorylation of NFP-H in perikarya or the axonal transport of NFP from perikarya to proximal axons is uniform in spinal motor neurons but variable in spinal ganglion cells.     

Aluminum Alters the Electrophoretic Properties of Neurofilament Proteins: Role of Phosphorylation State

Exposure of each of the three neurofilament proteins (NFPs) to AlCl3 resulted in their failure to migrate into sodium dodecyl sulfate (SDS)-containing gels. This effect was dependent on length of incubation (minimum, 2 h) and AlCl3 concentrations (minimum, 50 microM) and was not reversed by 20% SDS, 6 M urea, freeze-thawing, boiling, or extensive dialysis. The migration of vimentin and glial fibrillary acidic protein was not affected by AlCl3. The high-molecular-weight neurofilament subunit (NF-H) entered SDS-containing gels after exposure to aluminum lactate but migrated aberrantly as a long high-molecular-weight streak. Migration of the 160-kDa alpha-chymotryptic cleavage product of NF-H, which contains the higher phosphorylated tail domain, was also prevented from migrating into SDS-containing gels by AlCl3. Dephosphorylation of NF-H and the middle-molecular-weight neurofilament subunit (NF-M) eliminated these effects on gel migration. EDTA, EGTA, MgCl2, CaCl2, or FeCl3 had no effect on NF-H or NF-M migration; furthermore, preincubation with, or simultaneous exposure to, CaCl2 or FeCl3 did not alter the effect of AlCl3. One interpretation of these results is that Al3+ interacts with phosphate groups on extensively phosphorylated C-terminal sidearms of NFPs, resulting in intermolecular cross-linking. These findings demonstrate a direct effect of aluminum on NFPs and provide a possible mechanism for neurofilament accumulation in perikarya during aluminum intoxication.     

Posttranslational modification of a neurofilament protein during axoplasmic transport: implications for regional specialization of CNS axons

The possibility that proteins are modified during axoplasmic transport in central nervous system axons was examined by analyzing neurofilament proteins (200,000, 140,000, and 70,000 mol wt) along the mouse primary optic pathway (optic nerve and optic tract). The major neurofilament proteins (NFPs) exhibited considerable microheterogeneity. At least three forms of the “ 140,000” neurofilament protein differing in molecular weight by SDS PAGE (140,000-145,000 mol wt) were identified. The “140,000” proteins, and their counterparts in purified neurofilament preparations, displayed similar isoelectric points and the same peptide maps. The “140,000” NFPs exhibited regional heterogeneity when consecutive segments of the optic pathway were separately examined on polyacrylamide gels. Two major species (145,000 and 140,000 mol wt) were present along the entire length of the optic pathway. The third protein (143,000 mol wt) was absent proximally but became increasingly prominent in distal segments. After intravitreal injection of [(3)H]proline, newly synthesized radiolabeled proteins in the “140,000” mol wt region entered proximal mouse retinal ganglion cell (RGC) axons as two major species corresponding to the 145,000 and 14,000 mol wt NFPs observed on stained gels. When transported NFPs reached more distal axonal regions (30 d postinjection or longer), a 143,000 mol wt protein appeared that was similar in isoelectric point and peptide map to the 145,000 and 140,000 mol wt species. The results suggest that (a) the composition of CNS neurofilaments, particularly the “140,000” component, is more complex than previously recognized, that (b) retinal ganglion cell axons display regional differentiation with respect to these cytoskeletal proteins, and that (c) structural heterogeneity of “140,000” NFPs arises, at least in part, from posttranslational modification during axoplasmic transport. When excised but intact optic pathways were incubated in vitro at pH 7.4, a 143,000 NFP was rapidly formed by a calcium-dependent enzymatic process active at endogenous calcium levels. Changes in major proteins other than those in the 145,000-140,000 mol wt region were minimal. In optic pathways from mice injected intravitreally with L-[(3)H]proline, tritiated 143,000 mol wt NFP formed rapidly in vitro if radioactively labeled NFPs were present in distal RGC axonal regions (31 d postinjection). By contrast, no 143,000 mol wt NFP was generated if radioactively labeled NFPs were present proximally in RGC axons (6 d postinjection). The enzymatic process that generates 143,000 mol wt NFP in vitro, therefore, appears to have a nonuniform distribution along the RGC axons. The foregoing results and other observations, including the accompanying report (J. Cell Biol., 1982, 94:159-164), imply that CNS axons may be regionally specialized with respect to structure and function.     

Enriched Environment Protects the Optic Nerve from Early Diabetes-Induced Damage in Adult Rats

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Axoglial alterations of the distal (close to the chiasm) optic nerve (ON) could be the first structural change of the visual pathway in streptozotocin (STZ)-induced diabetes in rats. We analyzed the effect of environmental enrichment on axoglial alterations of the ON provoked by experimental diabetes. For this purpose, three days after vehicle or STZ injection, animals were housed in enriched environment (EE) or remained in a standard environment (SE) for 6 weeks. Anterograde transport, retinal morphology, optic nerve axons (toluidine blue staining and phosphorylated neurofilament heavy immunoreactivity), microglia/macrophages (ionized calcium binding adaptor molecule 1 (Iba-1) immunoreactivity), astrocyte reactivity (glial fibrillary acid protein-immunostaining), myelin (myelin basic protein immunoreactivity), ultrastructure, and brain derived neurotrophic factor (BDNF) levels were assessed in non-diabetic and diabetic animals housed in SE or EE. No differences in retinal morphology or retinal ganglion cell number were observed among groups. EE housing which did not affect the STZ-induced weight loss and hyperglycemia, prevented a decrease in the anterograde transport from the retina to the superior colliculus, ON axon number, and phosphorylated neurofilament heavy immunoreactivity. Moreover, EE housing prevented an increase in Iba-1 immunoreactivity, and astrocyte reactivity, as well as ultrastructural myelin alterations in the ON distal portion at early stages of diabetes. In addition, EE housing avoided a decrease in BDNF levels induced by experimental diabetes. These results suggest that EE induced neuroprotection in the diabetic visual pathway.     

The structure and development of the rat retina: an immunofluorescence microscopical study using antibodies specific for intermediate filament proteins

Rat retina structure was studied between embryonic day 14 and adult with antibodies specific for vimentin, glial fibrillary acidic protein (GFA) and the proteins of the neurofilament triplet. Vimentin could be detected in radial processes throughout the retina at all stages studied. These processes are believed to correspond, in the developing retina, to ventriculocytes, and in the mature retina to Müller cells. They could not normally be stained with any of the other intermediate filament antibodies employed here. We did find, however, that some older albino rats possessed GFA staining in addition to vimentin in these processes. Since we never saw such staining in the retinae of mature non-albino rats, and the retinae of older albino rats often showed signs of degeneration, we concluded that such GFA expression was most likely pathological. Neurofilament protein-positive processes were first detectable at embryonic day 15 1/2 in the inner regions of the retina, and corresponded to the axons of retinal ganglion cells. Such processes were equivalently displayed with antibodies to 68 K and 145 K protein, but were negative with 200 K protein. Some 68 K and 145 K positive fibers could also be decorated with vimentin antibody at this stage, though at later stages this was not the case. At later development stages more 68 K and 145 K neurofilament positive processes appeared, and after the first post-natal week progressively more of such processes became in addition 200 K positive, so that almost all neurofilament positive fibers in the adult stained for all three proteins. Such fibers, in the mature retina corresponded to 68 K and 145 K positive optic nerve fibers, and the processes of neurones in the inner plexiform layer. All fibers in the mature optic nerve fiber layer, but not all of those in the inner plexiform layer were stainable with 200 K antibodies. At 4 days post-natal we were able to detect 68 K and 145 K protein positive profiles in the outer regions of the developing retina, the prospective outer plexiform layer. Such profiles were always in addition vimentin positive, but negative for 200 K protein. During further development such profiles became ordered into a well defined layer and from about post-natal day 13 all of them began to acquire 200 K protein. They could be identified as the processes of horizontal cells. They continued to express vimentin in addition to the three triplet proteins in the adult, a so far unprecedented situation. We were able to detect neurofilament staining in the mature retina only in the above described regions, the inner and outer nuclear layer and the photoreceptor processes being completely free of staining. GFA was first detected in short processes adjacent to the inner limiting membrane which penetrated the optic nerve fiber layer. Such profiles were first detectable in the eye of the newborn animal, and were invariably identically stainable with vimentin at this age. These profiles could be stained with both vimentin and GFA at all later stages examined, although GFA staining became very much stronger than vimentin staining in some profiles in the adult. The results presented here are discussed in terms of development of the different retinal cell types.     

The neurofilament middle molecular mass subunit carboxyl-terminal tail domains is essential for the radial growth and cytoskeletal architecture of axons but not for regulating neurofilament transport rate

The phosphorylated carboxyl-terminal "tail" domains of the neurofilament (NF) subunits, NF heavy (NF-H) and NF medium (NF-M) subunits, have been proposed to regulate axon radial growth, neurofilament spacing, and neurofilament transport rate, but direct in vivo evidence is lacking. Because deletion of the tail domain of NF-H did not alter these axonal properties (Rao, M.V., M.L. Garcia, Y. Miyazaki, T. Gotow, A. Yuan, S. Mattina, C.M. Ward, N.S. Calcutt, Y. Uchiyama, R.A. Nixon, and D.W. Cleveland. 2002. J. Cell Biol. 158:681-693), we investigated possible functions of the NF-M tail domain by constructing NF-M tail-deleted (NF-MtailDelta) mutant mice using an embryonic stem cell-mediated "gene knockin" approach that preserves normal ratios of the three neurofilament subunits. Mutant NF-MtailDelta mice exhibited severely inhibited radial growth of both motor and sensory axons. Caliber reduction was accompanied by reduced spacing between neurofilaments and loss of long cross-bridges with no change in neurofilament protein content. These observations define distinctive functions of the NF-M tail in regulating axon caliber by modulating the organization of the neurofilament network within axons. Surprisingly, the average rate of axonal transport of neurofilaments was unaltered despite these substantial effects on axon morphology. These results demonstrate that NF-M tail-mediated interactions of neurofilaments, independent of NF transport rate, are critical determinants of the size and cytoskeletal architecture of axons, and are mediated, in part, by the highly phosphorylated tail domain of NF-M.     

Sertoli and Leydig cells of the human testis express neurofilament triplet proteins

 Using RT-PCR, western blot and enzyme and fluorescence immunocytochemical techniques, the three isoforms of neurofilament proteins (NFPs), namely NF-L (NFP-68 kDa), NF-M (NFP-160 kDa) and NF-H (NFP-200 kDa) were found in Sertoli and Leydig cells of human testes. RT-PCR showed specific for the three NFP fragments in testicular tissue, in isolated seminiferous tubules and in isolated Leydig cells. In protein preparations from the same testicular components, western blot analysis detected bands with molecular weights characteristic for NF-H, NF-M and NF-L. Application of immunofluorescence and immunoenzyme methods on cryostat and paraffin sections resulted in differences in the staining pattern in Sertoli cells and Leydig cells. In these cells, the NFPs showed predominantly a perinuclear location from which bundles emerge that were directed towards the basal, apical and lateral extensions of the Sertoli cells as well as the periphery of Leydig cells. NF-H coexists with vimentin-type filaments as seen by dual staining and staining of conseccutive serial sections of material embedded in paraffin. In Sertoli cells, vimentin and NF-H showed distinct dynamic changes depending on the stage of spermatogenesis and some structural variations of seminiferous tubules. Although in some tubules both vimentin and NF-H immunoreactivity was present at high levels, in the Sertoli cells from most individuals an inverse relationship in the staining intensity of vimentin and NF-H was observed. The strongest NF-H immunoreactivity was detected in Sertoli cells associated with stage 3 spermatids, whereas vimentin immunoreactivity was most abundant in association with stage 5 spermatids. The leydig cells did not show functional changes of the NFP immunoreactivity. The results obtained provide new evidence for the heterogeneous phenotype of human Sertoli cells and raise the question of their exact nature and origin. Accepted: 17 November 1998     

Differential turnover of phosphate groups on neurofilament subunits in mammalian neurons in vivo

The phosphorylation and dephosphorylation of specific proteins was demonstrated directly in the intact vertebrate nervous system in vivo. By exploiting the neurons' ability to segregate a select group of cytoskeletal proteins from most other phosphorylated constituents of the cell by axoplasmic transport, we were able to examine the dynamics of phosphate turnover on neurofilament proteins in mouse retinal ganglion cell neurons simultaneously labeled with [32P]orthophosphate and [3H]proline in vivo. Three [3H]proline-labeled neurofilament protein (NFP) subunits, designated H (160-200 kDa), M (135-145 kDa), and L (68-70 kDa), entered optic axons in a mole:mole ratio similar to that of isolated axonal neurofilaments, supporting the notion that newly synthesized NFPs are transported along axons as assembled neurofilaments. NFP subunits incorporated high levels of 32P before reaching axonal sites at the level of the optic nerve. As neurofilaments were transported along axons, however, many initially incorporated [32P]phosphate groups were removed. Loss of these phosphate groups occurred to a different extent on each subunit. A minimum of 50-60 and 35-40% of the labeled phosphate groups was removed in a 5-day period from the L and M subunits, respectively. By contrast, the H subunit exhibited relatively little or no phosphate turnover during the same period. Dephosphorylation of L in axons is accompanied by a decrease in its net state of phosphorylation; changes in the phosphorylation state of H and M, however, also reflect ongoing addition of phosphates to these polypeptides during axonal transport (Nixon, R.A., Lewis, S.E., and Marotta, C.A. (1986) J. Neurosci., in press). The possibility is raised that dynamic rearrangements of phosphate topography on NFPs represent a mechanism to coordinate interactions of neurofilaments with other proteins as these elements are transported and incorporated into the stationary cytoskeleton along retinal ganglion cell axons.     

High- and medium-molecular-weight neurofilament proteins define specific neuron types in the guinea-pig enteric nervous system

Previous studies have demonstrated that neurofilament proteins are expressed by type II neurons in the enteric plexuses of a range of species from mouse to human. However, two previous studies have failed to reveal this association in the guinea-pig. Furthermore, immunohistochemistry for neurofilaments has revealed neurons with a single axon and spiny dendrites in human and pig but this morphology has not been described in the guinea-pig or other species. We have used antibodies against high- and medium-weight neurofilament proteins (NF-H and NF-M) to re-examine enteric neurons in the guinea-pig. NF-H immunoreactivity occurred in all type II neurons (identified by their IB4 binding) but these neurons were never NF-M-immunoreactive. On the other hand, 17% of myenteric neurons expressed NF-M. Many of these were uni-axonal neurons with spiny dendrites and nitric oxide synthase (NOS) immunoreactivity. NOS immunoreactivity occurred in surface expansions of the cytoplasm that did not contain neurofilament immunoreactivity. Thus, because of their NOS immunoreactivity, spiny neurons had the appearance of type I neurons. This indicates that the apparent morphologies and the morphological classifications of these neurons are dependent on the methods used to reveal them. We conclude that spiny type I NOS-immunoreactive neurons have similar morphologies in human and guinea-pig and that many of these are inhibitory motor neurons. Both type II and neuropeptide-Y-immunoreactive neurons in the submucosal ganglia exhibit NF-H immunoreactivity. NF-M has been observed in nerve fibres, but not in nerve cell bodies, in the submucosa. This work was supported by a grant from the National Health and Medical Council of Australia (grant number 400020).     

Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons

Catecholaminergic cells are transiently present during development of the fetal murine bowel. These transient catecholaminergic (TC) cells appear at Day E10, but by Day E13 can no longer be detected. In order to evaluate the hypothesis that these cells are the precursors of enteric neurons, we investigated the possibilities that TC cells coexpress neuronal and catecholaminergic markers, that they can be found along the presumed path followed by crest-derived cells migrating to the gut, and that they are proliferating. TC cells were identified immunocytochemically using polyclonal or monoclonal antibodies to tyrosine hydroxylase (TH). At Day E9.5, TH-immunoreactive cells were observed to be present along the wall of the primordial esophagus in lines that extended from the developing nodose ganglia down to the boundary of the stomach. At Day E9.5, TC cells were absent from the remaining foregut. These lines of esophageal TH-immunoreactive cells became continuous with similar cells in the wall of the stomach and duodenum on Day E10. Coincident expression of neurofilament immunoreactivity was seen in all of the esophageal TH-immunoreactive cells present at Day E9.5, as well as in the entire set of esophageal and lower enteric TH-immunoreactive cells present at Day E10 (or later); moreover, at Days E9.5 and E10, all of the neurofilament-immunoreactive cells in the esophagus, stomach, or duodenum were also TH-immunoreactive. In contrast, neurofilament immunoreactivity was not expressed by the endodermally derived pancreatic duct and islet cells, which were also TH-immunoreactive; nor could expression of neurofilament immunoreactivity be detected in the TH-immunoreactive cells of the nodose ganglia. It was not until Day E11 that neurofilament-immunoreactive cells, which did not coexpress TH immunoreactivity (the definitive phenotype of enteric neurons) began to appear in the gut. Vagal axons reached as far distally as the nodose ganglion on Day E9.5, the esophagogastric junction on Day E10, and did not enter the stomach until Day E11. When the vagus nerves reached their level, the TH-immunoreactive cells in the wall of the esophagus came to lie among the nerve fibers. TH-immunoreactive cells are thus present on the pathway ultimately followed by the vagus nerves, but they develop before vagal fibers reach their level. The vagal TH-immunoreactive cells, therefore, are probably not initially migrating on vagal fibers, but appear instead to be overtaken by the descending vagus nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

青年猫和老年猫视神经超微结构观察比较

目的探讨青年猫和老年猫视神经年龄相关的形态学变化及可能造成的生理影响。方法取4只青年猫(2-3岁,2-2.5kg)和4只老年猫(10-13岁,2.5-3.5kg)颅内相对应部分视神经,制作横向半薄切片和超薄切片,半薄切片用甲苯胺蓝硼砂溶液染色,光镜观察、测量视神经的直径(不含外层神经膜);超薄切片标本用醋酸和柠檬酸铅染色,电镜观察、计数视神经纤维密度、测量视神经纤维外径D(含髓鞘)和内径d(不含髓鞘),按一定分级范围算出各种直径的纤维及各种d/D比值的纤维所占百分比,分别画出直方图,对实验结果进行统计学分析并绘制纤维直径谱。结果与青年猫相比,老年猫视神经直径显著增大(P0.05);纤维数量显著下降(P0.05)。纤维直径谱分析结果显示,青、老年猫纤维直径分布范围相似,但老年猫纤维的峰直径及纤维平均直径比青年猫的显著减小(P0.05),老年猫视神经纤维的d/D比值亦明显降低。另外,老年猫视神经中部分轴突肿胀,髓鞘疏松、结构紊乱,板层脱离、空泡化,有的轴索髓鞘溶解。结论在衰老过程中,老年猫视神经纤维丢失,纤维直径减小,d/D比值下降,以及纤维髓鞘的松散解体,这些变化均可能导致视神经纤维对视觉信息的传导速度减慢,是老年个体视觉分析速度下降的重要原因。     

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.     

Multiple fates of newly synthesized neurofilament proteins: evidence for a stationary neurofilament network distributed nonuniformly along axons of retinal ganglion cell neurons

We have studied the fate of neurofilament proteins (NFPs) in mouse retinal ganglion cell (RGC) neurons from 1 to 180 d after synthesis and examined the proximal-to-distal distribution of the newly synthesized 70-, 140-, and 200-kD subunits along RGC axons relative to the distribution of neurofilaments. Improved methodology for intravitreal delivery of [3H]proline enabled us to quantitate changes in the accumulation and subsequent decline of radiolabeled NFP subunits at various postinjection intervals and, for the first time, to estimate the steady state levels of NFPs in different pools within axons. Two pools of newly synthesized triplet NFPs were distinguished based on their kinetics of disappearance from a 9-mm "axonal window" comprising the optic nerve and tract and their temporal-spatial distribution pattern along axons. The first pool disappeared exponentially between 17 and 45 d after injection with a half-life of 20 d. Its radiolabeled wavefront advanced along axons at 0.5-0.7 mm/d before reaching the distal end of the axonal window at 17 d, indicating that this loss represented the exit of neurofilament proteins composing the slowest phase of axoplasmic transport (SCa or group V) from axons. About 32% of the total pool of radiolabeled neurofilament proteins, however, remained in axons after 45 d and disappeared exponentially at a much slower rate (t 1/2 = 55 d). This second NFP pool assumed a nonuniform distribution along axons that was characterized proximally to distally by a 2.5-fold gradient of increasing radioactivity. This distribution pattern did not change between 45 and 180 d indicating that neurofilament proteins in the second pool constitute a relatively stationary structure in axons. Based on the relative radioactivities and residence time (or turnover) of each neurofilament pool in axons, we estimate that, in the steady state, more neurofilament proteins in mouse RGC axons may be stationary than are undergoing continuous slow axoplasmic transport. This conclusion was supported by biochemical analyses of total NFP content and by electron microscopic morphometric studies of neurofilament distribution along RGC axons. The 70-, 140-, and 200-kD subunits displayed a 2.5-fold proximal to distal gradient of increasing content along RGC axons. Neurofilaments were more numerous at distal axonal levels, paralleling the increased content of NFP.(ABSTRACT TRUNCATED AT 400 WORDS)     

An antigen present in the Drosophila central nervous system only during embryonic and metamorphic stages.

We report here about an antigen that is expressed in the central nervous system (CNS) of Drosophila only during the embryonic and metamorphic stages. In Drosophila, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropil of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and neutral pI in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the CNS is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immunoreactivity in the CNS presumably reflect dynamic changes of an extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis.     

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.     

Xefiltin,a Xenopus laevis neuronal intermediate filament protein,is expressed in actively growing optic axons during development and regeneration

Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as α-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, α-internexin and gefiltin. α-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 811–824, 1997     

Phosphorylation of bovine neurofilament proteins by protein kinase FA (glycogen synthase kinase 3).

A highly purified preparation of protein kinase FA (where FA is the activating factor for phosphatase 1)/glycogen synthase kinase 3 from rabbit muscle readily phosphorylated bovine neurofilaments. All three neurofilament proteins, the high, middle, and low molecular proteins (NF-H, NF-M, and NF-L), were phosphorylated when intact filaments were incubated with the kinase. Experiments with individual proteins showed that NF-M was the best substrate. At protein concentrations of 0.13 mg/ml, the initial rate of NF-M phosphorylation was 30% of that observed for glycogen synthase. Km values were 0.24 mg/ml (7 x 10(-7) M tetramer) for glycogen synthase and 0.10 mg/ml (5 x 10(-7) M dimer) for NF-M. Vmax values were 0.36 mumol/min/mg for glycogen synthase and 0.035 mumol/min/mg for NF-M. Dephosphorylated NF-M was phosphorylated only half as much as native NF-M; this is consistent with the known substrate specificity of the kinase. The possible involvement of FA/GSK-3 in the phosphorylation of neurofilaments in vivo is discussed.     

The Differential Role of Protein Kinase C Isozyme in the Rapid Induction of Neurofilament Phosphorylation by Nerve Growth Factor and Phorbol Esters in PC12 Cells

We examined the short-term regulation of the phosphorylation of the mid-sized neurofilament subunit (NF-M) by kinases which were activated in rat pheochromocytoma (PC12) cells by nerve growth factor (NGF) and/or 12-O-tetradecanoylphorbol 13-acetate (TPA). We found that NGF and TPA, alone or in combination, increased (a) the incorporation of [32P]Pi into NF-M and (b) the rate of conversion of NF-M from a poorly phosphorylated to a more highly phosphorylated form. This was not due to increased synthesis of NF-M, because NGF alone did not increase NF-M synthesis and TPA alone or TPA and NGF together inhibited the synthesis of NF-M. Further, an increase in calcium/phospholipid-dependent kinase (PKC) activity resulting from the treatment of PC12 cells with NGF and TPA was observed concomitant with the increased phosphorylation of NF-M. This PKC activity was determined to be derived from the PKC alpha and PKC beta isozymes. Finally, when PC12 cells were rendered PKC-deficient by treatment with 1 muM TPA for 24 h, NGF maintained the ability to induce an increase in NF-M phosphorylation, though not to the level attained in cells which were not PKC-deficient. These data suggest that NGF with or without TPA stimulates NF-M phosphorylation as a result of a complex series of events which include PKC-independent and PKC-dependent pathways.     

Characterization of Additional Casein Kinase I Sites in the C-Terminal "Tail" Region of Chicken and Rat Neurofilament-M

Abstract: In previous studies we have identified Ser⁵⁰², Ser⁵²⁸, and Ser⁵³⁴ as target sites in chicken neurofilament middle molecular mass protein (NF-M) for casein kinase I (CKI) in vitro and have shown that these sites are also phosphorylated in vivo. We now make use of a combination of molecular biological and protein chemical techniques to show that two additional in vivo phosphorylation sites in chicken NF-M, Ser⁴⁶⁴ and Ser⁴⁷¹, can also be phosphorylated by CKI in vitro. These two sites are conserved in higher vertebrate NF-M molecules, and recombinant protein constructs containing the homologous rat NF-M peptides can be phosphorylated by CKI in vitro, suggesting that phosphorylation of these sites is conserved at least in higher vertebrates. The two new sites are adjacent to a conserved peptide sequence (VEE-IIEET-V) found once in higher vertebrate NF-M molecules and twice in lamprey NF-180. Variants of this sequence are also found in neurofilament low and high molecular mass proteins (NF-L and NF-H) and α-internexin, and in mammalian NF-L are known to be associated with in vivo phosphorylation sites. We speculate that CKI phosphorylation in general, and these sites in particular, may be important in neurofilament function.