Slow posttranslational modification of a neurofilament protein.

The synthesis and subsequent modification of neurofilament (NF) polypeptides has been examined in pulse-chase experiments, using cultured chick spinal cord neurons. Fluorography of the [35S]methionine-labeled cytoskeletal proteins, after separation by two-dimensional gel electrophoresis, revealed that (a) the mid-size chicken NF protein, NF-M160, is synthesized as a smaller and more basic precursor, NF-M130; (b) beginning approximately 8 h after translation, NF-M130 slowly and continuously becomes larger and more acidic, attaining the size and charge of NF-M160 16 or more h later, and undergoing no further change in mobility for many days thereafter; and (c) in contrast, the low molecular weight NF protein, NF-L, is synthesized as such, and undergoes no subsequent change in apparent size or charge. Additional experiments provided evidence that the conversion of NF-M130 to NF-M160 is due, at least in part, to phosphorylation: (a) Incubation of similar cultures in 32PO4 resulted in incorporation into NF-M160 and transitional forms, but not into NF-M130. (b) An antiserum to NF-M160 was found by immunoblot analysis to bind strongly to untreated NF-M160, but poorly to phosphatase-treated NF-M160, and not at all to NF-M130. It has already been demonstrated (Bennett, G. S., S. J. Tapscott, C. DiLullo, and H. Holtzer, 1984, Brain Res., 304:291-302) that this anti-NF-M160 fails to stain the soma of motor neurons in sections of chick spinal cord, but detects an increasing gradient of immunoreactivity in the proximal axons. These results, together with the known kinetics of axoplasmic transport of NF, suggest that the mid-size chicken NF protein is synthesized as NF-M130 and is extensively modified, at least in part by phosphorylation, to become NF-M160 during transport along proximal neurites. Once maximally modified, NF-M160 undergoes no further net change during transport along distal neurites.

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro.

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.

Amino acid incorporation into rat brain proteins during spreading cortical depression.

Unilateral spreading cortical depression was elicited by applying potassium chloride solutions to the dura of conscious, freely moving rats. Incorporation of (3)H-leucine into soluble cortical proteins was decreased in the depressed hemisphere relative to the control side, while soluble brainstem proteins from both sides had the same specific activity. Various subfractions of soluble cortical proteins were affected to equal degrees.

Different proteins associated with 10-nanometer filaments in cultured chick neurons and nonneuronal cells.

A protein of molecular size 180 kilodaltons is associated with 10-nanometer filaments in neurons and is immunologically distinct from smaller putative neurofilament subunits and from 10-nanometer filament proteins in nonneuronal cells, such as myotubes and fibroblasts. Neurons do not contain vimentin, the major filament protein in many other cells, including the nonneuronal cells in cultures of neural tissue.

Immunoglobulin-like domains define the nerve growth factor binding site of the TrkA receptor.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system. NGF binds with high affinity to the extracellular region of the tyrosine kinase receptor TrkA. This domain comprises leucine and cysteine rich motifs, followed by two immunoglobulin like (Ig-like) domains. We describe the expression and purification of recombinant Ig-like domains. Fluorescence and circular dichroism spectroscopy show that the protein is folded into a compact globular structure and contains mainly beta-sheet secondary structure. Recombinant protein binds to NGF and can inhibit NGF bioactivity both in vitro and in vivo.

Identification of four alternatively spliced isoforms of chicken casein kinase I alpha that are all expressed in diverse cell types.

Casein kinase I (CKI) is a family of widely expressed protein kinases. It is previously shown in mammalian tissues that CKIalpha exists as two or three alternatively spliced isoforms (Rowles et al.,1991; Zhang et al., 1996; Kuret et al., 1997). We now report that four alternatively spliced isoforms of CKIalpha are expressed in many chicken cells and tissues. A partial cDNA clone was isolated from a chicken brain library, using a probe derived from a bovine CKIalpha cDNA. The translated sequence of this clone was 100% identical to the bovine homolog containing the 'L' insert, with the addition of 12 amino acids just before the C terminus that had previously been reported in human and Xenopus CKIalpa. After completing the missing portion of the coding sequence by 5' RACE (rapid amplification of cDNA ends), full-length cDNA was PCR amplified from chicken brain cDNA, yielding four different products. These were cloned and sequenced and found to correspond to the four CKIalpha isoforms: CKIalpha, CKIalphaL, CKIalphaS and CKILalphaLS, where 'S' is the insert consisting of the 12 human/Xenopus C-terminal amino acids. Using reverse transcription and polymerase chain reaction (Rt-PCR), it was shown that the four isoforms are all expressed in neurons, fibroblasts and several tissues. This represents the first demonstration that four splice variants exist and are all expressed in a single type of cell.

Neurogenic cutaneous vasodilatation and plasma extravasation in diabetic rats: effect of insulin and nerve growth factor.

1. Neurogenic vasoactive responses in rat skin were investigated following 8 weeks of streptozotocin-induced diabetes to determine the effect of diabetes and of treatment with insulin and nerve growth factor (NGF) treatment. 2. Diabetic rats were divided into three groups: untreated; insulin (4 IU day(-1) by s.c. implant weeks 4-8) treated; Nerve Growth Factor, NGF, (0.2 mg kg(-1) three times weekly, weeks 4-8) treated. A fourth group served as a non-diabetic control. 3. Electrical stimulation of the saphenous nerve (10 V, 2 Hz, 1 ms for 30 s) increased blood flow in the ipsilateral paw skin, as measured by laser Doppler flowmetry. The peak increase was similar between groups, but the time taken for flow to return to a steady baseline was significantly (P < 0.01) reduced in untreated diabetic rats, when compared with non-diabetic controls, but not significantly reduced in the insulin- or NGF-treated diabetic groups. 4. A second stimulation of the saphenous nerve (10 V, 2 Hz, 1 ms for 5 min) produced plasma extravasation, measured by the extravascular accumulation of 125I-albumin, in the skin. Plasma extravasation was significantly attenuated (P < 0.001) in the untreated diabetic group, but not the insulin-treated group, compared to non-diabetic controls. Plasma extravasation was present, though reduced, in the NGF-treated group. 5. Plasma extravasation induced by intradermal injections of substance P with and without CGRP was similar in all groups indicating no decrease in vascular responsiveness to exogenously applied neuropeptides. The results suggest that release of neuropeptides is diminished in diabetes and that treatment with either insulin or NGF can restore neurogenic microvascular vasoactive responses towards normal.

Neurogenic oedema and vasodilatation: effect of a selective neuronal NO inhibitor.

The effects of a neuronal nitric oxide synthase (nNOS) inhibitor, 1-(2-trifluoromethylphenyl)imidazole (TRIM) on rat sensory saphenous nerve-induced neurogenic inflammation were investigated. TRIM (50 mg kg-1, i.p.), but not 2-trifluoromethylphenol (TRIMPOH) which lacks nNOS inhibitory activity, inhibited neurogenic oedema by 55.8 +/- 6.5% (n = 6, p < 0.05). The effect of TRIM was partially reversed by L-arginine (100 mg kg-1, i.v., p < 0.01). TRIM also caused a reduction (p < 0.05) in neurogenic vasodilatation but had no effect on neuropeptide responses induced by substance P + CGRP. Topically applied TRIM (100 microliters of 150-250 mg ml-1) inhibited neurogenic oedema (p < 0.01). Thus, use of this recently described nNOS inhibitor has provided new evidence to further the hypothesis that nNOS plays a role in modulating sensory nerve-mediated neurogenic inflammation.

Characterization of a neurofilament-associated kinase that phosphorylates the middle molecular mass component of chicken neurofilaments.

We have examined the properties of a chicken neurofilament (NF) kinase partially purified from NF-enriched preparations. This kinase cosediments with NFs following extraction with Triton X-100 and can be separated in an active form from NFs by treatment with 0.8 M KCl. Sequential chromatography of the salt extract on DEAE-cellulose and phosphocellulose results in an approximately 500-fold increase in specific activity over endogenous NF preparations as measured by 32P-incorporation into the middle molecular mass component of NFs (NF-M). The kinase is Mg(2+)-dependent, second messenger-independent and inhibited by high concentrations of heparin. It shows selectivity for NF-M and evidence is presented that the kinase phosphorylates NF-M solely in the tail domain. The kinase can also phosphorylate the microtubule-associated proteins tau and MAP2 as well as mammalian NF-M, all of which share putative phosphorylation sequences with chicken NF-M.

Differential binding of antibodies against the neurofilament triplet proteins in different avian neurons.

Using monospecific antisera against each of the three chicken neurofilament (NF) proteins, NF70, NF160 and NF180, the distribution of each of these proteins in several types of neurons was examined by immunohistochemistry. Striking differences were observed in the relative staining by the three antibodies when the soma of different types of neurons were compared, and also when the soma of some neurons were compared with their axons. Both the soma and axons of dorsal root sensory neurons were brightly stained by each of the antisera. The soma of spinal cord ventral horn neurons, however, were stained only by A-NF70 and A-NF180, not by A-NF160. The axons of these neurons were uniformly stained by A-NF70 and A-NF180, while only gradually becoming NF160-positive over the first several hundred microns. The lack of staining by A-NF160 was also observed in many neuronal soma in cultures of dissociated spinal cord cells. The soma and dendrites of adult cerebellar Purkinje cells were weakly stained by A-NF70 and A-NF180 and not at all by A-NF160, but both A-NF70 and A-NF180 yielded prominent staining of immature Purkinje cells and dendrites. These results suggest that the three NF proteins may be unequally distributed within the soma and processes of different types of neurons and/or may be subject to regionally selective modification.

Pyruvate carboxylase: an astrocyte-specific enzyme implicated in the replenishment of amino acid neurotransmitter pools.

Pyruvate carboxylase is the predominant anaplerotic enzyme in CNS tissues, and thus provides for net utilization of glucose to generate citric acid cycle intermediates such as alpha-ketoglutarate and malate for replenishment of the neurotransmitter pools of glutamate, GABA and aspartate. Studies reported in this paper involving immunocytochemical and biochemical techniques demonstrate: (1) the enzyme is localized in astrocytes as visualized by immunofluorescence in sections of cerebellum and (2) the enzyme activity in astrocyte-enriched populations is 3 X higher than in granule cell-enriched populations isolated from the cerebellum; similarly activity in different synaptosomal preparations parallels that for glutamine synthetase. We conclude from these results that the enzyme pyruvate carboxylase is an astrocyte-specific marker. This localization substantiates some recent hypotheses for astrocyte functions, including CO2 fixation in the CNS and the replenishment of citric acid cycle intermediates by astrocytes as precursors for amino acid neurotransmitter pools.

Identification of Ser-Pro and Thr-Pro phosphorylation sites in chicken neurofilament-M tail domain.

The tail domain of the midsize chicken neurofilament polypeptide (NF-M) contains several different types of Ser-Pro and Thr-Pro putative phosphorylation sites. We determined which of these sites are actually phosphorylated in vivo. Chick sensory neuron cultures were incubated in [32P]phosphate, and the cytoskeletal fraction was mixed with a neurofilament fraction prepared from adult chicken brain. NF-M was purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and digested with chymotrypsin, and two large fragments were isolated. These were individually cleaved with trypsin, endoprotease Lys-C, or endoprotease Glu-C, and peptides separated by two-dimensional high-voltage electrophoresis and thin-layer chromatography. 32P-labeled phosphopeptides were eluted from the cellulose plates and subjected to microsequencing and mass spectometry. We found that of 21 potential Ser-Pro and Thr-Pro phosphoacceptor sites, at least 20 are phosphorylated in vivo: all four Lys-Ser-Pro sites and at least 16 of the 17 Lys-Xaa-Xaa-Ser/Thr-Pro repeats. In addition, a novel Ser-Pro site in the extreme carboxy terminus is phosphorylated. This site, which has no proximal Lys residue, is also found in mammalian NF-M, but has not been reported to be phosphorylated. Together with three casein kinase I sites we have found recently in the acidic amino-terminal segment of the tail, a total of 24 or 25 Ser and Thr phosphoacceptor sites have now been located in the chicken NF-M tail.

Transient expression of a neurofilament protein by replicating neuroepithelial cells of the embryonic chick brain.

An immunohistochemical survey was carried out on frozen sections of the early embryonic chick brain between 1 and 6 days of incubation, with antisera to the three neurofilament proteins (NF-L, NF-M, NF-H). Large numbers of replicating neuroepithelial cells were found to express one of these proteins, NF-M, generations before the existence of any postmitotic neuroblasts (Days 1-2 1/2 of incubation). NF-L and NF-H could not be detected. Not all primordial brain regions contained NF-M-positive cells, but in those that did, every cell was positive. These regions included the dorsal forebrain, optic vesicles, and dorsal hindbrain, but not the dorsal midbrain. All cells in all regions of the cephalic neural tube contained vimentin, whether or not they also contained NF-M. This NF-M expression was transient in the sense that later generations of these NF-M-positive neuroepithelial cells became NF-M negative, before finally giving rise to some descendents that ultimately express all three NF proteins. This transient NF-M expression was found in certain other cells of early embryos, including cardiac myoblasts. The identity of the component in these early neural and nonneural tissues, that bound the antibody, was demonstrated to be identical to adult brain NF-M by one- and two-dimensional immunoblots. These findings demonstrate an unusual kind of biochemical heterogeneity among neuroepithelial cells, and they are relevant to considerations regarding lineage analysis and lineage "markers" in the vertebrate central nervous system.

Expression of a neurofilament protein by the precursors of a subpopulation of ventral spinal cord neurons.

The expression of neurofilament proteins (NF-H, NF-M, and NF-L) in replicating neuroepithelial cells and postmitotic neuroblasts in the embryonic chick trunk neural tube was examined by immunohistochemistry. Anti-NF-M, in particular, resulted in bright staining of some mitotic cells, which were found to be strictly localized to a midventral and an extreme dorsal position in the neural tube. Those in the midventral position were observed with greatest frequency during Days 3 and 4 of incubation and became increasingly rare thereafter. During the same period of time, and in the same small ventral region, NF-M-positive interphase cells, presumably migrating postmitotic neuroblasts, were also present. In contrast, NF-L-positive mitotic cells were rarely seen. NF-L-positive migrating and differentiating neuroblasts were observed throughout the ventral half of the neural tube except in the midventral area containing NF-M-positive mitotic cells and NF-M-positive migrating neuroblasts. These results, together with known temporal and spatial patterns of neurogenesis in the spinal cord, suggest that the expression of NF-L and NF-M, in the form recognized by our antibodies, may not be initiated coordinately, or even in the same sequence, in different types of neuroblasts, and that only the immediate precursors of a specific subpopulation of ventral spinal cord neurons begin expressing NF-M in the terminal cell cycle. In addition, the NF-M-positive mitotic cells, when observed in anaphase and telophase, had NF-M-positive material associated with both emerging daughter cells and the migrating neuroblasts were frequently found in closely associated pairs, consistent with the suggestion that these precursor cells undergo a symmetrical terminal division to yield two daughter postmitotic neuroblasts.

Lithium chloride alters cytoskeletal organization in growing, but not mature, cultured chick sensory neurons.

Biochemical analysis indicates that lithium ion (Li+) has deleterious effects on the metabolism of at least two elements of the cytoskeleton in cultured chick dorsal root ganglia (DRG) neurons. Phosphorylation of newly synthesized middle molecular mass neurofilament polypeptide (NF-M) is inhibited by 10-25 mM LiCl, and tubulin (Tb) synthesized in the presence of Li+ is subject to rapid degradation. These Li(+)-induced metabolic abnormalities are accompanied by alterations in cellular and cytoskeletal morphology. Treatment of cultures having vigorously growing neurites with 25 mM LiCl results in the cessation of net neurite growth, without causing neurite retraction. Indirect immunofluorescence reveals that in these cultures Li+ provokes an aggregation of NF protein into a dense knot in the cell body/proximal neurite region. The knots contain accumulations of all three NF polypeptides and electron microscopic observation demonstrates that the knots contain intact, but disorganized, filaments. Both the inhibition of neurite outgrowth and NF collapse are reversible. Tubulin and intact microtubules are redistributed in immature cultures treated with Li+ insofar as they are excluded from the NF knots. Neurons in established cultures (e.g., 7 days and beyond) fail to show any difference between Li+ treatment and control conditions in the morphology of the cytoskeletal elements examined.

Expression and phosphorylation of the mid-sized neurofilament protein NF-M during chick spinal cord neurogenesis.

The middle molecular weight polypeptide of neurofilaments (NF-M) is modified posttranslationally by extensive phosphorylation. This modification is slow in mature neurons, requiring approximately 24-48 hr for completion and probably occurs outside of the cell soma (Bennett and DiLullo: J Cell Biol 100:1799, 1985c). Thus, NF-M synthesis and phosphorylation are separate events both temporally and spatially. Although it is known that NF-M is among the earliest neuron-specific gene products to be expressed during nervous system development, it is not known what the temporal relationship is between the initiation of NF-M translation and its phosphorylation. To address this question, we have produced an antiserum against the dephosphorylated form of NF-M (NF-M130) and have used this antiserum, together with a previously characterized antiserum against completely phosphorylated NF-M (NF-M160), in an immunohistochemical examination of neurogenesis and the initial period of neuronal differentiation in chick spinal cord. We found that 1) nonphosphorylated and partially phosphorylated NF-M cannot be detected prior to the completion of the terminal mitosis; 2) most postmitotic neuroblasts begin expressing NF-M as they commence migration, but do not contain the completely phosphorylated polypeptide until some time after completion of migration; and 3) those precursor cells of a subpopulation of neuroblasts that begin expressing completely phosphorylated NF-M during their terminal cell cycle (Bennett and DiLullo: Dev Biol 107:94, 1985a) contain no detectable nonphosphorylated or partially phosphorylated NF-M. These cells probably complete the phosphorylation step more rapidly than do mature neurons.

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons.

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut.(ABSTRACT TRUNCATED AT 400 WORDS)

Defective expression of neurofilament protein subunits in hereditary hypotrophic axonopathy of quail.

Hereditary hypotrophic axonopathy is an inherited, neuronal cytoskeletal disease of the "quiver" mutant quail (Quv). Nerve tissue pathology is characterized by axonal hypotrophy associated with neurofilament (NF) deficiency in the central and peripheral nervous system. To elucidate the biochemical mechanism of this disease, we examined the in vivo expression of NF triplet protein subunits and the assembly state thereof in neuronal cell bodies in adult Quv and controls. Gel electrophoresis and Western blot analysis indicated that low, middle and high molecular mass NF subunits were markedly deficient in the brain, cervical spinal cord and sciatic nerve of Quv. Immunohistochemically, the spinal cord of Quv had no immunoreactive products corresponding to low molecular mass NF. However, middle and high molecular mass NF antisera stained few axons in the white matter and bound to ventral horn cell bodies, which in the controls were not labeled. Furthermore, a 130-kDa subunit likely to be a non- or hypophosphorylated form of middle molecular mass NF was localized in neuronal cell bodies with considerably stronger intensity than those in the controls. Ultrastructurally, intermediate filaments were not seen in such neurons; instead amorphous matrix was increased between clusters of granular endoplasmic reticulum and in the peripheral cytoplasmic areas. Degenerative changes of the neurons were very rare. We hypothesize that the deficiency of NFs in Quv results from an alteration of filament assembly caused by defective expression of low molecular mass NF. This mutant presents direct evidence for the importance of NFs in achieving and/or maintaining normal axon caliber and provides a novel system for further studies on NF expression, metabolism and function.

Immunofluorescent visualization of 100 A filaments in different cultured chick embryo cell types.

Antibody prepared against the 55,000 dalton subunit of reconstituted chick gizzard 100 A filaments (anti-G55K) bound to the 100 A filaments of chick smooth muscle, cardiac muscle, and skeletal muscle cells, and to the 100 A filaments of Schwann cells and satellite glial cells of the peripheral nervous system. Anti-G55K did not bind to replicating presumptive myoblasts, fibroblasts, chondroblasts, pigment cells, neurons, or to central nervous system glial cells. This contrasted with the wider range of binding of antibody to the 58,000 dalton subunit of chick fibroblast 100 A filaments (anti-F58K) which bound to the 100 A filaments of all cell types examined except hepatocytes and skin epithelial cells. Anti-G55K) staining revealed a morphologically distinct distribution of 100 A filaments in the three types of muscle cells. Spindle shaped smooth muscle cells exhibited dense fluorescent staining near the poles of the cells, and also exhibited unique patches of fluorescent material after cytochalasin B and Colcemid treatment. In myotubes, the fluorescence was limited to longitudinal bundles of filaments between the striated myofibrils. Cardiac cells contained uniformly distributed fine filaments. Lastly, smooth muscle cells in various phases of mitosis bound the anti-G55K, whereas replicating presumptive skeletal myoblasts failed to bind the anti-G55K.