Ballooned neurons in select neurodegenerative diseases contain phosphorylated neurofilament epitopes

Summary Ballooned neurons are histological features of several neurodegenerative diseases of the central nervous system. We describe the immunocytochemical staining of ballooned neurons in Pick's disease, unclassified dementia, corticonigral degeneration, pigment-spheroid degeneration and Alzheimer's disease. In all of these conditions the ballooned neurons contain phosphorylated epitopes recognized by monoclonal antibodies to neurofilaments, but not epitopes unique to Alzheimer neurofibrillary tangles and Pick bodies. The morphological features and immunohistochemical properties of ballooned neurons in these disorders bear resemblance to swollen neurons produced by neurotoxins that impair axoplasmic transport of neurofilaments. This finding, by analogy, suggests that impaired axoplasmic transport of neurofilaments may be a common mechanism in various dementing neurodegenerative diseases.     

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.     

The axon reaction in motor and sensory neurones of mice studied by a monoclonal antibody marker of neurofilament protein

Following unilateral sciatic nerve crush in mice, changes in the neurofilament content of neuronal perikarya were studied, using a monoclonal antibody to neurofilament protein (RT97). In the spinal cord, anterior horn motor neurones, normally unstained, showed a positive staining reaction with immunoperoxidase on the operated side. This reaction was short lived and maximal on the 11th post-operative day. In spinal ganglia, the proportion of positively staining sensory neurones showed an earlier but otherwise similar increase. In both cases, the response was well defined and contrasted with the changes on Nissl staining, which were markedly different in the two populations of neurones. In the nerve crush region, although regenerating axons were visible with silver staining only 5 days post-operatively, neurofilament protein was not demonstrated in these axons until several days later, after the peak perikaryal increase. These results suggest that an increase in perikaryal neurofilament protein is a consistent and quantifiable event following distal axon trauma, possibly indicating either synthesis of protein subunits or repolymerization of neurofilaments prior to their transport distally down the regenerating axons. The findings may be useful in identifying neurones with distal axon lesions in experimental and other neuropathological material.     

Characterization of a panel of neurofilament antibodies recognizing N-terminal epitopes.

Peptides corresponding to sequences from the amino-terminal "head" regions of the low, middle, and high molecular weight neurofilament proteins (NF-L, NF-M, and NF-H) were synthesized by a modification of the Merrifield solid-phase method, and a panel of polyclonal antibodies to these epitopes were prepared in rabbits by the injection of synthetic peptides conjugated to the carrier protein keyhole limpet hemocyanin (KLH). An additional, monoclonal antibody recognizing both glial fibrillary acidic protein (GFAP) and vimentin was also produced, by fusion of cells of the mouse myeloma line NS-1 with spleen cells from a mouse immunized with cytoskeletal extracts. Antibody specificities were confirmed by a combination of Western blotting against cytoskeletal extracts and immunofluorescence using both rat brain sections and fibroblasts transfected with fully encoding cDNAs for each neurofilament protein, driven by viral promoters.     

Alteration of the neurofilament sidearm and its relation to neurofilament compaction occurring with traumatic axonal injury

Traumatic injury evokes two characteristic forms of focal axonal injury, one of which involves focal perturbation of axolemmal permeability associated with rapid compaction of the underlying axonal neurofilament lattice and microtubular loss. In this process, the neurofilament sidearms have been the subject of intense scrutiny in relation to their role in this NF compaction, with the suggestion that the sidearms, thought to maintain interfilament distance, are proteolytically cleaved and degraded at the time of injury. The current communication addresses the fate of the NF sidearms in such injured axons. Adult cats were subjected to moderate/severe fluid percussion brain injury after intrathecal administration of horseradish peroxidase (HRP). This tracer, excluded by the intact axolemma of uninjured axons, was used to recognize injured axons via HRP intra-axonal uptake/flooding with HRP. Animals were perfused and processed for light microscopic and electron microscopic study of both HRP-containing and non-HRP-containing axons from the same field. HRP-containing axons consistently displayed evidence of traumatically-induced (NF) cytoskeletal collapse. Electron micrographs of HRP-containing axons as well as uninjured, non-HRP-containing axons from the same fields were videographically captured, digitized, enlarged and analysed for NF sidearm length and NF density. HRP-containing axons were found to have increased NF density. Surprisingly, this increased NF density occurred despite the retention of the NF sidearms, which now, however, were reduced in height in comparison to the non-HRP-containing uninjured axons. These observations are not consistent with previously published reports suggesting that overt proteolytic degradation of sidearms was responsible for NF compaction. Based on our findings, we suggest that the NF compaction associated with traumatically-induced axolemmal permeability changes may have its genesis in more subtle sidearm modification, perhaps involving a change in phosphorylation state.     

Serum antibodies to neurofilament antigens in patients with neurological and other diseases and in healthy controls

A total of 529 sera from patients with a wide variety of neurological and non-neurological diseases, and 101 sera from healthy control subjects, were examined by indirect immunofluorescence for the presence of autoantibodies to neurofilament antigens. Antibodies were found in approximately 50% of sera from patients with spongiform encephalopathies, 15-30% of sera from patients with other neurological and non-neurological diseases, and 7% of sera from healthy controls. The antigenic stimulus to these autoantibodies in very diverse disease processes is unknown, but as presently assayed, they are not of sufficient specificity to be useful as an aid to clinical diagnosis.     

Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: in vivo and in vitro study with monoclonal antibodies

Neurofilament phosphorylation in rat nervous system development was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes in tissue sections and in primary dissociated cultures. The antibodies either decorated neurofilaments shortly after their appearance or after a considerable delay (from 4 to 9 days in vivo and from 12 to 27 days in vitro), thus suggesting the existence of at least two classes of phosphorylated epitopes. With most antibodies there was a good correlation between in vivo and in vitro findings as to the early or late appearance of phosphorylated epitopes. Monoclonal NE14 was the main exception in that immunoreactivity with this antibody was present in 1-day cultures, while it only occurred 4 days after the first appearance of neurofilaments in vivo. The effect of phosphorylation on neurofilament structure and function remains to be determined. Neurofilament expression is an early phenomenon in ontogeny coinciding with neuronal differentiation. It is possible that late phosphorylation events may stabilize the axonal cytoskeleton following the massive loss of axons that occurs in several fiber tracts during late fetal and neonatal life.     

Sulfated glucuronyl glycolipids reacting with anti-myelin-associated glycoprotein monoclonal antibodies including IgM paraproteins in neuropathy: species distribution and partial characterization of epitopes

It was recently established that anti-myelin associated glycoprotein (MAG) IgM paraproteins associated with neuropathy and a substantial number of experimentally produced rat and mouse monoclonal antibodies that react with MAG (e.g. HNK-1) also bind to some sulfated glucuronic acid-containing sphingoglycolipids of human peripheral nerve. A species study revealed that these glycolipids could be detected readily by TLC overlay experiments in the acidic glycolipid fractions from human, monkey, bovine, cat and dog peripheral nerve. The glycolipids were also present in the nerves of rat, mouse, rabbit, guinea pig and chicken, but their concentration was about an order of magnitude lower. These antigenic glycolipids were present in the purified myelin fraction from cat nerve, but their level was not enriched over that in whole homogenate. Partial characterization of the epitopes in the glycolipids was accomplished by comparing binding of the human and experimental monoclonal antibodies to sulfated glucuronyl paragloboside (SGPG), to the desulfated lipid (GPG), and to the methyl ester of the desulfated lipid (MeGPG). All of the human, mouse and rat antibodies reacted with the intact SGPG, but none exhibited binding to MeGPG indicating that either the sulfate or the free carboxyl group on SGPG was required for reactivity. Five out of 11 human IgM paraproteins retained partial and variable reactivity with GPG showing that the sulfate was not absolutely required for binding, while the other 6 did not react with GPG. These results demonstrate idiotypic heterogeneity among the IgM paraproteins. Only 1 of 14 monoclonal antibodies produced experimentally in mice or rats retained reactivity with GPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of human tissue-type plasminogen activator with monoclonal antibodies: mapping of epitopes and binding sites for fibrin and lysine.

The study defines interactions between human tissue-type plasminogen activator (t-PA) and 21 mouse monoclonal antibodies (mAb). Characterization includes epitope distribution, reactivity of different forms of t-PA with antibodies, and modification of t-PA function by antibody binding. Eighteen antibodies are directed against t-PA A-chain. These antibodies recognize four distinct epitopes (A, B, C, D) and one partially overlapping epitope (D'). The remaining three antibodies are directed against two different epitopes (E, F) on catalytically active t-PA B-chain. A-chain reactive antibodies do not bind to the reduced form of t-PA, while B-chain reactive antibodies bind to reduced and deglycosylated t-PA forms. The latter antibodies associate more tightly with sc t-PA than with tc t-PA and have a higher affinity for t-PA-PAI 1 complex as compared to free t-PA. The analysis of functional effects of antibodies reveals that antibodies directed against all above defined epitopes inhibit interactions between t-PA and fibrin: a) binding of t-PA to fibrin, b) fibrinolytic activity of t-PA, and c) fibrin activation of sc t-PA amidolytic activity. The observations support the assumption that several sites of t-PA are involved in fibrin binding and that fibrin-bound t-PA is closely surrounded by the fibrin mesh. Many antibodies quench also binding of t-PA to lysine-Sepharose. Experiments with free, non-fixed lysine confirm strong competition between lysine and mAb 16 and 18, directed against epitope A, and mAb 29, binding to epitope F.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hereditary hypotrophic axonopathy with neurofilament deficiency in a mutant strain of the Japanese quail

Summary A primary axonal disease affecting the central and peripheral nervous system was discovered in a new mutant strain of the Japanese quail, named quiver (Quv). Quv showed significantly smaller cross-sectional areas of the cervical spinal cord and the optic and sciatic nerves, when compared with controls by light microscopic morphometry. In the cervical spinal cord, electron microscopic morphometry indicated that myelinated axons in Quv were significantly smaller in size than in controls, though greater in density. The axonal circularity was not significantly different from that of controls. Electron microscopically and immunohistochemically, neurofilaments were not detected in the axons or neuronal cell bodies. Axons in Quv were composed mainly of microtubules, which were increased in number in relation to the axonal size. From these findings, the lesions observed in Quv were regarded as axonal hypotrophy (growth arrest or retardation) due to altered neurofilament expression.     

IgG monoclonal proteins from patients with axonal peripheral neuropathies bind to different epitopes of the 68 kDa neurofilament protein.

We describe three patients with a sensorimotor axonal polyneuropathy and an IgG M-protein that binds to a 68 kDa axonal protein identified as the low molecular weight neurofilament protein (NF-L). The immunological studies revealed that the M-proteins have different target epitopes: one is phosphorylated and the other two are nonphosphorylated. One of the nonphosphorylated epitopes is common to other intermediate filaments, such as desmin and vimentin.     

Family of human neuronal external surface epitopes defined by Torpedo monoclonal antibodies.

We are employing a library of monoclonal antibodies (MAbs) that were made to Torpedo cholinergic synaptosomes to identify conserved, physiologically vital epitopes of the neuronal surface. Our particular interest is in those epitopes that are present on some but not all neurons. In the present study we screened this library on different cell lines, the neuronal cell lines PC12, NG108, MC-IXC, and SY5Y, and the endocrine cell lines GH-3 and HIT. Of these cell lines, only SY5Y cells bind MAbs that define neuronal surface subsets. Utilizing its parent cell line, SK-N-SH, we verified that six MAbs, Tor 25, Tor 103, Tor 190, Tor 201, Tor 219, and Tor 233, bind the external neuronal surface. The cytolocalization of all six MAbs is very similar: the membrane of the cell body and its processes are finely outlined in a punctate distribution. Western blot analyses of Torpedo electric organ homogenates, a highly enriched source of antigenic material, revealed that each MAb identifies multiple polypeptides, two of which have the relative mobilities of 180 kD and 67 kD. In a screen of peripheral nerves from cases of amyotrophic lateral sclerosis (ALS), we found that all these MAbs revealed surface alterations; some displayed a decrease in binding, while others displayed an increase. The combined data provide evidence that these epitopes belong to an important, complex family of polypeptides of the external neuronal surface.     

Developmental expression of GD3 and polysialogangliosides in embryonic chicken nervous tissue reacting with monoclonal antiganglioside antibodies

The appearance and developmental distribution of GD3 and polysialogangliosides was studied immunohistochemically by means of the monoclonal mouse antibodies AbR24 and Q211. Cryostat sections of chicken embryos from 1 to 5 days (E1-E5) of incubation (Hamburger and Hamilton, stages 9-26) were prepared. GD3, detected by AbR24, is expressed on E2 by proliferating neuroepithelial cells of all regions of the developing brain and spinal cord and by migrating neural crest cells. As development continues, GD3 drops to a much lower level in postmitotic neurons, but is still strongly expressed by cells of the mitotically active germinal zones. In non-neural tissues the antigen was found in much lower concentrations than in the neuroectoderm, with the exception of very early, heavily proliferating endodermal and mesodermal epithelia. In contrast, the antigen(s) of the monoclonal mouse antibody Q211, identified on TLC-plates in polysialoganglioside fractions, was found to be specific for central and peripheral derivatives of the neural ectoderm. In the CNS, these polysialogangliosides are absent in the proliferating neural epithelium and appear for the first time on cells of the developing peripheral mantle layers at E2.5 (stage 17). Sections from all brain areas, retina, optic stalk, spinal cord, and peripheral nerves and ganglia of later stages (E4-5) showed that the antigen(s) of Q211 is expressed by neuronal perikarya and cell processes. The data strongly suggest that the developmental appearance of these polysialogangliosides is synchronized with the withdrawal of proliferating precursor cells from the mitotic cycle and differentiation to neurons.     

Staining with monoclonal antibodies to neurofilaments distinguishes between subpopulations of neurofibrillary tangles,between groups of axons and between groups of dendrites

Summary A new monoclonal antibody (mab) against neurofilaments is described (mab 1215) and its reactions compared with previously characterized mabs (BF10; RT97). Mab 1215 recognizes an epitope on the heavy neurofilament polypeptide (NF-H). In Alzheimer's disease, mab 1215 recognizes only a subpopulation of neurofibrillary tangles and stains a proportion of tangles in the hippocampus but none of those in the olfactory bulb. However, mabs RT97 and BF10 stain the majority of tangles in both brain areas. Of the three antibodies, only mab BF10 recognizes, specifically, axons of granular cells in the dentate gyrus of the hippocampus. Mab 1215 recognizes more dendrites in the pyramidal layer than either mab BF10 or mab RT97. Our observations indicate that neurofilaments are not identical in all axons and that, contrary to previous reports, NF-H is present in dendrites. The dendritic form of NF-H appears to be different from NF-H in axons and this could be due to differences in the state of phosphorylation of NF-H. We suggest that the finding that distinct subpopulations of tangles exist indicates that tangles are not static lesions. Further investigations into this possibility may illuminate the pathophysiology of Alzheimer's disease.     

Distribution of phosphate-independent MAP2 epitopes revealed with monoclonal antibodies in microwave-denatured human nervous system tissues

In contrast with results obtained in experimental animals, antibodies to microtubule associated protein-2 (MAP2) preferentially label abnormal structures in human nervous system tissue samples, but the normal sites at which MAP2 is expressed are not well-defined. To determine the distribution of MAP2 in the human central (CNS) and peripheral (PNS) nervous systems, we prepared monoclonal antibodies (MAbs) specific to MAP2, and compared the localization of this MAP in postmortem bovine and human tissues as well as in several human neural cell lines that express either neurofilament (NF) or glial filament (GF) proteins. Eight MAbs specific for phosphate-independent epitopes in bovine and human MAP2 were obtained, and those that performed well in tissues produced immunoreactivity confined to the somatodendritic domain of neurons in bovine and human CNS and PNS tissues. Other neural cells (e.g. astrocytes) did not express MAP2 immunoreactivity using these MAbs. Postmortem delays of less than 24 h prior to tissue denaturation did not affect the distribution of MAP2 immunoreactivity. However, microwave denaturation of these tissues preserved MAP2 immunoreactivity better than fixation with Bouin's solution or formalin. Microwave treatment also improved the immunoreactivity of several MAbs for NF and GF proteins. Finally, MAP2 was not detected in human neural cell lines that express NF (2) or GF (1) proteins. We conclude that microwave denaturation provides an effective means to preserve the immunoreactivity of normal human neuronal cytoskeletal proteins, and that this method of tissue denaturation allows the normal distribution of MAP2 to be defined in postmortem samples of human CNS and PNS tissues.     

Comparative binding of murine and human monoclonal antibodies reacting with myelin-associated glycoprotein to myelin and human lymphocytes

Human monoclonal IgM antibodies present in the blood of some patients with peripheral neuropathy and murine hybrid IgM antibodies C5 and C6, raised against myelin-associated glycoprotein, and HNK-1, raised against the human T cell line HSB-2, all bind to the carbohydrate moiety of myelin-associated glycoprotein. The relative avidity of the monoclonal antibodies was HNK-1 greater than C5/C6 much greater than human IgM, as determined in a competitive binding radioimmunoassay. HNK-1 bound myelin equally well at incubation temperatures between 4 degrees C and 37 degrees C; the human antibodies bound significantly only at 4 degrees C; and C6 bound best at 4 degrees C, less strongly at 20 degrees C and did not bind at 37 degrees C. All of the antibodies bound to a band corresponding to myelin-associated glycoprotein on immunoblots of human CNS myelin proteins in addition to several other antigens. Flow cytometric studies revealed that the murine but not the human antibodies bind to peripheral blood lymphocytes. Taken together, these data suggest that the antibodies probably recognize the same epitope but bind with different avidity.     

Neurophysin in the hypothalamo-neurohypophysial system. I. Production and characterization of monoclonal antibodies

Seven mouse monoclonal antibodies (IgGs) were produced against rat neurophysins (NPs). Three were specifically directed against vasopressin-associated NP (NP-AVP), and four were specific for oxytocin-associated NP (NP-OT). These specificities were observed in liquid phase assays, immunoblot, and immunoprecipitation experiments. Homozygous Brattleboro rat tissues and extracts, which do not contain vasopressin or NP-AVP, did not react with the anti-NP-AVP antibodies but reacted with high affinity to the anti-NP-OT antibodies. In immunoprecipitation assays the antibodies brought down the appropriate NPs as well as their precursor molecules synthesized in vivo with no detectable cross-reactivity. In solid phase assays where the antigens were presented in a different manner, there was a significant cross-reactivity of the anti-NP-AVP antibodies with NP-OT. The extent of this cross-reactivity in solid phase correlated with the cross-reactivities of the antibodies observed in immunocytochemical studies. These solid phase (and immunocytochemical) data demonstrated that liquid phase specificities and absorption controls of antibodies are inadequate to assess their immunocytochemical (solid phase) specificities. Posterior pituitary extracts from the mouse and frog, as well as purified NPs from the rat, cow, and human were studied for their cross-reactivities to two of the antibodies, PS 36 and PS 45. In liquid phase assays the anti-rat NP-OT antibody, PS 36, reacted only with rat and mouse NPs and did not cross-react with NPs from any of the other species. In contrast, the anti-rat NP-AVP antibody, PS 45, was cross-reactive across species lines including an NP-like antigen extracted from frog posterior pituitaries. Immunoblot staining with these antibodies showed heterogeneity of NP-AVP and NP-OT in the rat posterior pituitary. Analysis of the epitopes for PS 36 and PS 45 indicated the antigenic determinants were located near amino acid positions 80 to 81 in NP-OT and 75 to 86 in NP-AVP, respectively.     

First appearance of laminin in peripheral nerve, cerebral blood vessels and skeletal muscle of the rat embryo: Immunofluorescence study with laminin and neurofilament antisera

The formation of basal laminae in peripheral nerve was studied by immunofluorescence with laminin antisera in the rat embryo. Peripheral nerves were identified with neurofilament antisera in double labeled sections. In the adult rat perineurium and endoneurium were uniformly decorated by the antisera. Sensory neurons in posterior root ganglia were surrounded by a laminin positive basal lamina. Laminin immunoreactivity was first observed in posterior spinal roots on day 14. Anterior spinal roots and peripheral nerves remained laminin negative until day 17. The adult pattern (uniform decoration of endoneurium in large and small nerve trunks) was only observed on day 21. The formation of a basal lamina surrounding posterior root ganglion neurons was still not completed in 3-day-old rats. The only laminin positive structures in the brain and spinal cord were the external basal laminae and the blood vessels. The external basal lamina was present at all stages of development. In the spinal cord and brain stem vascular basal laminae were first identified with laminin antisera on day 14, in the diencephalon and telencephalon on day 15. Laminin immunoreactivity in the basal laminae surrounding myotubes was first observed on day 16.     

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.