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.     

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet. Of these nine antibodies, two stained motor neuron perikarya in the spinal cord, while the remaining seven antibodies were axon-specific in this location. Subpopulations of stained and unstained motor neurons were not observed. With all 12 antibodies, the staining pattern in the lumbar dorsal root ganglia and spinal cord remained unchanged following sciatic nerve crush and ligature. The findings suggest that, in the neurofilament, some phosphorylated epitopes are axon specific, while other phosphorylated epitopes are present in both axons and perikarya. Furthermore, they suggest that differences exist between neuronal populations as to the presence of phosphorylated epitopes in perikaryal neurofilaments. It remains to be seen whether phosphorylation events in perikarya and axons have similar or different effects on neurofilament structure and function.     

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.     

Transient expression of neurofilament protein without filament formation in purkinje cell development: Immunohistological and electron microscopic study of chicken cerebellum

Purkinje cell bodies and dendrites in the chick embryo stained intensely by immunofluorescence and by the Avidin-Biotin Complex (ABC) method using monoclonal and polyclonal antibodies to the neurofilament (NF) proteins. On day 20 (the day before hatching) NF immunoreactivity markedly decreased concomitant with the first appearance of NF-positive basket axons in the molecular and Purkinje cell layers. On postnatal day 3, as in mature avian cerebellum, NF-negative Purkinje cells were surrounded by NF-positive baskets. Cytoplasmic 10 nm filaments were not observed in immature and mature Purkinje cells by electron microscopy. Basket axons in the hen were packed with 10 nm filaments.     

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.     

Early and late appearance of neurofilament phosphorylation events in nerve regeneration

Neurofilament phosphorylation in regenerating rat sciatic nerve was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes of the 2 large polypeptides of the neurofilament protein triplet (NF 150K, NF 200K). One group of antibodies decorated axons early in the process. In fact, no differences were seen in double labeled sections between these antibodies and polyclonal neurofilament antibodies as to their reactivity with the distal stump of transected sciatic nerves. Another group stained axons after they had completed their elongation, i.e., after they had reached the distal part of the denervated sciatic nerve. In general, the epitopes recognized by antibodies in this group appeared more sensitive to phosphatase digestion as compared to the first group. Furthermore, there was a good correlation between the thickness of the regenerated axons and staining with these monoclonal antibodies. Thick axons (like those observed in normal nerves) were stained, while bundles of thin axons remained unstained. Monoclonal II32 stained regenerated axons in a remarkable segmental pattern. With this antibody, continuous decoration of the axons was still not observed 7 weeks after transection, the longest follow-up period in this study. We suggest that some neurofilament phosphorylation events may contribute to the stabilization of the axonal cytoskeleton and that abnormalities persist in regenerated axons as to the extent of neurofilament phosphorylation.     

Non-phosphorylated and phosphorylated neurofilaments in hypothyroid rat cerebellum

Purkinje cell baskets in hypothyroid rat cerebellum were studied with antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes. Compared to normal rats, Purkinje cell baskets were fewer in number and less developed in hypothyroid rat cerebellum. However, no differences were observed as to their immunoreactivity with monoclonal antibodies reacting with phosphorylated or non-phosphorylated neurofilament epitopes.     

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     

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.     

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.     

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     

Brain filament proteins in primary cultures derived from chick embryos early in development

Brain filament expression and neurofilament post-translational modifications (phosphorylations) were studied in primary cultures derived from whole 3–4 day chick embryos. After 2–3 days in culture, neurofilament-positive cells formed neuronal aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. Aggregates and neuritic bundles were stained with several monoclonal antibodies reacting with phosphorylated neurofilament epitopes. With two monoclonal antibodies reacting with phosphorylated forms of the high molecular weight neurofilament subunit, staining was only observed after 8 and 10 days in vitro. There was a major difference between rat and chicken with respect to astrocyte differentiation in culture. In chicken, the flat cells surrounding the neuronal aggregates remained constantly GFAP-negative throughout the whole experimental period (10 days). GFAP-positive cells were first observed within the neuronal aggregates on day 8 in vitro.     

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.     

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)     

Identities, antigenic determinants, and topographic distributions of neurofilament proteins in the nervous systems of adult frogs and tadpoles of Xenopus laevis

Three proteins with nominal molecular weights of 73 kDa (XNF-L), 175 kDa (XNF-M), and 205 kDa (XNF-H) were identified as putative neurofilament proteins in the nervous system of the frog, Xenopus laevis. These conclusions were based on four criteria: (1) these proteins were enriched in cytoskeletal preparations; (2) they reacted with a monoclonal antibody (anti-IFA) that cross-reacts with an epitope found in all intermediate filament proteins; (3) they cross-reacted with monoclonal antibodies directed against specific mammalian neurofilaments; and (4) antibodies that reacted with these proteins on Western blots specifically stained neurons in immunohistochemical analyses. The neurofilament proteins in Xenopus were antigenically similar, but not identical to mammalian neurofilament proteins. The principal difference was that four antibodies that reacted on Western blots with rat NF-H reacted with XNF-M in Xenopus. However, similarly to mammals, antibodies against phosphorylated XNF-M specifically labeled axons, whereas an antibody that reacted only with dephosphorylated epitopes on XNF-M specifically labeled neuronal cell bodies in immunohistochemistry. Three other antibodies that reacted equally well with untreated or alkaline-phosphatase-treated XNF-M or XNF-H proteins also showed axonally restricted staining in the adult Xenopus nervous system. An XNF-L (XC5D10) antibody was produced which stained axons and cell bodies equivalently throughout the adult Xenopus nervous system. By 3 days of development (stage 42; Xenopus tadpoles), antibodies to all three molecular weight forms of the frog neurofilament proteins detected specific neurons in the brainstem and spinal cord; and antibodies to phosphorylated and dephosphorylated epitopes on XNF-M could discriminate between axons and cell bodies in the rhombencephalon. The immunoreactivities of four antibodies directed at XNF-L, -M, or -H, which were unaffected by alkaline phosphatase treatment, differed significantly in their immunohistochemical staining patterns in adult vs. premetamorphic frogs.     

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.     

Expression of neurofilament proteins by Purkinje cells: ultrastructural immunolocalization with monoclonal antibodies

Purkinje cell bodies in rodent cerebellum have been shown to express neurofilament protein epitopes but neurofilaments are rarely seen in these perikarya by classical morphological approaches. In an attempt to solve this enigma the ultrastructural distribution of two neurofilament epitopes was studied by immunoelectron microscopy with two monoclonal antibodies (Mabs) of divergent specificity: one, Mab 04-7 recognized a phosphorylated epitope, the other, Mab 02-135 a non-phosphorylated epitope. Longitudinal filamentous elements were heavily labeled in basket cell axons and afferent nerve fibers with both Mabs. While Mab 04-7 was unreactive with Purkinje cells, the immunoperoxidase reaction product with Mab 02-135 was distributed in the form of patches with no filamentous substructure throughout the cytoplasm of these cells. The data complement the results of other immunocytochemical studies showing the presence of all 3 neurofilament constituent proteins in Purkinje cell bodies, and lead to the conclusion that in these perikarya the majority of neurofilament proteins are not assembled in the form of neurofilaments.     

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.     

Expression of neurofilament proteins in the hypertrophic granule cells of Lhermitte-Duclos disease: an explanation for the mass effect and the myelination of parallel fibers in the disease state

The expression of neurofilament (NF) proteins was examined in the surgical specimen from a 42-year-old woman with Lhermitte-Duclos disease. Hypertrophic granule cell neurons of the dysplastic tissues were reactive with monoclonal antibodies, including antibodies to each of the three human NF subunits. Furthermore, antibodies to dephosphorylation-dependent epitopes on NF proteins stained the cell bodies of hypertrophic granule cells, whereas antibodies to phosphorylation-dependent epitopes stained the enlarged and myelinated axons of the hypertrophic granule cells. Enzymatic dephosphorylation of this tissue abolished axonal staining with phosphorylation-dependent antibodies and uncovered determinants recognized by antibodies to the dephosphorylated state of NF proteins. The NF protein immunoreactivity of hypertrophic granule cells was indistinguishable from that of large, NF-rich neurons in control human cerebellum, suggesting that a normal pattern of expression and phosphorylation of NF proteins occurs in hypertrophic granule cells in Lhermitte-Duclos disease. An increased expression of NF proteins by cerebellar granule cells may account for many of the observed alterations of Lhermitte-Duclos disease, including the hypertrophy of the granule cells and enlargement of their axons, leading to the myelination of parallel fibers within the molecular layer of the cerebellum. Attention should now be directed at the underlying mechanisms which lead to the coordinated up-regulation of the three NF genes and whether or not additional gene products or cell types are altered in Lhermitte-Duclos disease.