Neurofilament phosphorylation in the axonless horizontal cells of rat retina

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

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

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

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

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

Neurofilament phosphorylation in neuronal perikarya following axotomy: a study of rat spinal cord with ventral and dorsal root transection

Rat spinal cord was stained by indirect immunofluorescence with 11 neurofilament monoclonal antibodies that recognize phosphorylated epitopes. All monoclonals were axon-specific in this location. The large motoneurons containing bundles of neurofilaments did not stain and the pattern remained unchanged after transection of the sciatic nerve in the thigh. With nine monoclonals, stained motoneurons were observed in the ventral horns 3 days, 5 days, 1 week, and 2 weeks after transection of the ventral roots close to the spinal cord. The abnormal motoneurons were typically scattered among normal (i.e., nonstained) cells. Even in animals showing the most severe reaction, the whole motoneuron population at the site of rhizotomy was not affected, stained and nonstained perikarya often coexisting side by side. Stained motoneurons were no longer observed 3 weeks after ventral root transection. Changes in neuronal immunoreactivity were also observed after dorsal root transection. However, a different population was affected, i.e., middle-sized neurons in dorsal horns and at the base of ventral horns. With two monoclonals (A9 and D21), cell bodies remained negative following all operations. It is concluded that axotomy in proximity of the cell body may induce certain neurofilament phosphorylation events in motor neuron perikarya, whereas other phosphorylation events remain confined to the axons under these experimental conditions. The absence of changes after transection of the sciatic nerve in the thigh suggests that neurofilament phosphorylation is a reaction to cell injury rather than a cellular event related to nerve regeneration.     

Neurofilament phosphorylation in peripheral nerve regeneration

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.     

Distribution of neurofilament antigens after axonal injury

Phosphorylated and nonphosphorylated epitopes of neurofilament (NF) proteins are distributed in different regions of individual neurons. Immunocytochemical methods, with monoclonal antibodies directed against phosphorylated and nonphosphorylated NF, demonstrated nonphosphorylated NF in perikarya and proximal axonal segments of neurons in dorsal root ganglia, while phosphorylated NF proteins were present in axons of these cells. The distribution of these epitopes of NF were examined at various times following injury of axons in the rat sciatic nerve. Between one and 21 days after crush of the proximal nerve, phosphorylated NF were present in neuronal perikarya. We have compared patterns of perikaryal immunoreactivity at one time point (three weeks) following a more distal crush or complete transection of the sciatic nerve. At this time period, following transection/ligation, phosphorylated NF immunoreactivity was not present in perikarya, but abnormal staining was observed after nerve crush. These altered distributions of phosphorylated epitopes of NF are of interest because several recent reports have indicated that similar, but not identical, abnormal staining patterns occur in human neurological diseases, including Alzheimer's disease and Parkinson's disease. In accord with previous studies, this investigation indicates that one response of neurons to injury, or to disease, is an abnormal distribution of phosphorylated epitopes of NF proteins.     

Phosphorylation of neurofilaments is altered in aluminium intoxication

Summary A series of monoclonal antibodies that distinguish phosphorylated and nonphosphorylated neurofilament (NF) epitopes was used to immunostain brain stem neurons from control rabbits and from rabbits chronically intoxicated with Aluminium (Al). In controls, none of the monoclonal antibodies to phosphorylated NF stained the perikaryon of neurons. In contrast, in animals treated with Al, all neuronal perikarya containing Al-induced neurofilament bundles (NB) and some lacking well-formed NB immunoreacted with two of the five antibodies to phosphorylated NF. Axons were stained by all five antibodies to phosphorylated NF in both control and Al-treated animals. A broadly reacting monoclonal antibody to a nonphosphorylated NF epitopes immunoreacted with neuronal cell bodies, dendrites and axons in control and Al-intoxicated animals regardless of the presence of Al-induced NB. Staining of Al-induced NB with one of the antibodies to phosphorylated NF was greatly diminished after treatment of sections with trypsin and phosphatase. It is concluded that NF which compose the Al-induced NB have different immunocytochemical characteristics from those of the NF present in the perkaryon of normal neurons. It is likely that, contrary to normal perikaryal NF, NF of Al-induced NB are phosphorylated. Moreover, phosphorylation of NF of Al-induced NB is probably abnormal, since NF of Al-induced NB have immunostaining characteristics different from NF of normal axons. Al-induced NB may result from abnormal phosphorylation of NF in the perikaryon, preventing normal axonal transport of these structures.Supported by grants from the National Institutes of Health NS 14509 and AG 00795     

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

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

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

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

A study of infantile motor neuron disease with neurofilament and ubiquitin immunocytochemistry

We report a patient with infantile motor neuron disease who had pathologic findings consistent with multisystem degeneration. Although the muscle showed denervation atrophy and spinal anterior horn cells showed either atrophy or ballooning degeneration consistent with lower motor neuron disease, the infant was hypertonic and spastic. Degenerative changes were also detected in the dorsal root ganglia, cerebellum, and thalamus. Immunohistochemical studies showed a paucity of neurofilament (NF) staining in the corticospinal tract and accumulation of phosphorylated NF in ballooned neurons. Antibodies to ubiquitin immunostained ballooned neurons in the dorsal root ganglia, anterior horns, and thalamus. Accumulation of ubiquitinated and phosphorylated NF epitopes in degenerating neurons suggest that basic abnormalities in the neuronal cytoskeleton may be instrumental in the pathogenesis of this disorder.     

Phosphorylation of neurofilaments is altered in amyotrophic lateral sclerosis

We used a library of monoclonal antibodies (Mab) that distinguish phosphorylated (P+) and non-phosphorylated (P-) neurofilament (NF) epitopes to examine phosphorylation of NF in lower motor neurons of patients with amyotrophic lateral sclerosis (ALS), of neurologically normal controls of different ages, and of patients with central chromatolysis due to injuries to motor root axons. Monoclonal antibodies directed to P+ NF immunostained five to ten times more neuronal perikarya in ALS than in age-matched controls. Spheroids, which are NF containing axonal enlargements, found in significantly greater number in proximal axons in ALS, were also intensely immunostained with Mab to P+ NF. Moreover, anterior root axons in five of eleven cases of ALS reacted only with the Mab to P+ NF, while both P- and P+ NF were present in motor roots from controls. In control groups, the number of neuronal perikarya and spheroids that immunoreacted with the Mab to P+ NF increased moderately with age. Chromatolytic lower motor neurons were recognized by Mab to P+ NF. Our results show that the process of phosphorylation is altered in ALS. We propose that phosphorylation of NF in ALS occurs prematurely and that it is more likely to be associated with an impairment of NF transport than to be part of a chromatolytic reaction of lower motor neurons.     

Normal neuronal cell bodies of the nucleus tractus mesencephalici nervi trigemini react with antibodies against phosphorylated epitopes on neurofilaments

Reactivity to antibodies directed against phosphorylated neurofilament epitopes is normally present in axons. Phosphorylated neurofilament immunoreactivity is not generally found in neuronal perikarya, except in abnormal states such as Alzheimer's disease. We found that cytoplasm of neurons of the nucleus tractus mesencephalici nervi trigemini in normal mice and rats reacts with monoclonal antibodies against phosphorylated epitopes on neurofilaments. This suggests either that phosphorylated epitopes on neurofilaments are localized in the perikarya of some normal neurons or that the antibodies that were used (SMI 31 and SMI 34) recognize more than phosphorylated epitopes.     

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

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

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.     

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

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

Axonal regeneration in old multiple sclerosis plaques

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

Neurofilament Phosphorylation is Increased in Ventral Horn Neurons of Neonatal Rat Spinal Cord Exposed to Cerebrospinal Fluid from patients with Amyotrophic Lateral Sclerosis

Aberrant neurofilament (NF) phosphorylation in the soma of the ventral horn neurons of neo-natal rat spinal cord is observed following exposure to cerebrospinal fluid (CSF) of patients suffering from Amyotrophic Lateral Sclerosis (ALS). CSF samples from ALS and non-ALS neurological patients were injected into the spinal subarachnoid space of 3 day old rat pups. After 48 h, sections of spinal cords were stained for the presence of phosphorylated NF epitopes with SMI-31 antibody. The number of neuronal soma staining with this antibody in the ventral and dorsal horns sides of the spinal cord was counted. There was a significant 3-fold increase in the number of soma stained with SMI-31 antibody in the ventral horns of rat spinal cords exposed to CSF of patients with ALS compared to cords from rats exposed to CSF of non-ALS patients and those which were not exposed to any CSF samples. Such an increase in staining of neuronal soma was not observed in the dorsal horns. Hyperphosphorylation of neuronal soma suggests an initial stage of degenerative changes occurring in the motor (ventral horn) neurons following exposure to circulating factor(s) in the CSF of patients with ALS.     

Spatial and temporal expression of phosphorylated and non-phosphorylated forms of neurofilament proteins in the developing nervous system of Xenopus laevis

Immunocytochemical studies of developing Xenopus laevis embryos and tadpoles (stages 12 1/2 to 46) were performed using a panel of 11 monoclonal antibodies to phosphorylated and non-phosphorylated forms of the neurofilament proteins. These included nine antibodies to the middle molecular weight neurofilament protein (XNF-M, 175 kDa), and two additional antibodies to non-phosphorylated forms of the other two neurofilament proteins (XNF-L, 73 kDa; XNF-H, 205 kDa). The developmental expression of XNF-M, XNF-L and XNF-H, and the progressive phosphorylation of XNF-M in the rhombencephalon, spinal cord, and optic nerve were studied using these antibodies. In the spinal cord and rhombencephalon, non-phosphorylated forms of XNF-M were initially detected during neural tube stages (stages 22-26), one day before XNF-L and XNF-H at early tadpole stages (stage 35/36). In the eye, XNF-M was observed initially during tailbud stages (stage 29/30), but neither XNF-L nor XNF-H was seen even by stage 46 (swimming tadpole). The phosphorylation of XNF-M occurred over a protracted period of several days, both in the neural tube and visual system, and could be divided into four phases. (1) When initially expressed, XNF-M was hypophosphorylated. This was indicated by the early immunostaining of axons and cell bodies with antibodies to dephosphorylated epitopes on XNF-M and by the absence of staining with antibodies to phosphorylated epitopes. (2) After a short timelag (3-9 h) axons were stained by some, but not all antibodies to phosphorylated epitopes. (3) Approximately one day later, all antibodies to phosphorylated epitopes stained the relevant axons. However, XNF-M was not yet fully phosphorylated, as indicated by the continued staining of these axons with antibodies to dephosphorylated epitopes of XNF-M. (4) Two to 3 days after the initial expression of XNF-M, dephosphorylated epitopes disappeared from the axons, establishing the adult pattern. During development, the most heavily phosphorylated neurofilament proteins present at a given stage were found first in distal regions of the axons and progressed gradually toward the neuronal perikarya as development proceeded. This gradient of phosphorylation, established early within the axon, suggests that neurofilaments in the axons mature from their distal ends toward the cell body, a process which may be regulated by local factors within the axons themselves. The similarity of the basic features of NF-M phosphorylation in mammalian, avian, and amphibian axons underscores the importance of this phenomenon for the development of a mature axon.     

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)     

Regulation of aberrant neurofilament phosphorylation in neuronal perikarya. III. Alterations following single and continuous beta, beta'-iminodipropionitrile administrations.

beta,beta'-Iminodipropionitrile (IDPN) administration produces giant neurofilament-filled axonal swellings in the first proximal internodes of large myelinated sensory and motor fibers without any accompanying axonal degeneration. In the present study, we asked whether proximal giant axonal swellings are sufficient to elicit aberrant neurofilament (NF) phosphorylation in neuronal perikarya. Rats were given a single intraperitoneal (i.p.) injection of IDPN (2 g/kg) followed by IDPN (0.1%) in the drinking water (continuous IDPN exposure) or tap water (single IDPN exposure) for two days to 7 weeks. Immunoreactivity to phosphorylated NF (pNF) epitopes (using monoclonal antibodies 6-17 and 7-05) was observed in L4 and L5 dorsal root ganglia (DRG) neurons beginning between one and 5 days, corresponding to the development of proximal giant axonal swellings. Quantitation of DRG neurons demonstrated maximal numbers of immunoreactive cell bodies to pNF epitopes (46-51%) by one week. The number of immunostained DRG cells was maintained in animals given continuous IDPN exposure, but declined significantly (P less than 0.001) in rats given a single injection of IDPN to 26 +/- 0.80% and 6 +/- 0.04% at 3 and 5 weeks, respectively. Ventral and dorsal root fibers, which undergo axonal atrophy distal to axonal swellings, showed intense immunoreactivity to pNF epitopes and a marked reduction or a complete lack of immunostaining to antibody 2-135 (directed against non-phosphorylated NF epitopes); pretreatment with alkaline phosphatase reversed this staining pattern. In a separate study, a similar alkaline phosphatase-sensitive lack of staining to antibody 2-135 was also observed in atrophic motor fibers in the DRG 4 weeks following nerve crush. It is suggested that aberrant NF phosphorylation in DRG neuronal cell bodies from IDPN-treated rats arises secondarily to an alteration in a retrogradely transported 'trophic' signal(s) to the neuron due to the presence of giant axonal swellings. Furthermore, pNFs in atrophic axons may correspond to stationary or slowly moving NFs in the axoplasm.