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N H Sternberger

Publications and source records attributed to N H Sternberger.

At least 37 records · Page 2Linked to original sources
PubMed1986

Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I.

Calmodulin-dependent protein kinase II (CaM kinase II) is associated with microtubule preparations and phosphorylates several endogenous proteins including microtubule-associated protein 2, tubulin, and an 80,000-dalton protein doublet (pp80). We now report that pp80 is identical to synapsin I by all criteria studied including molecular weight, isoelectric point, phosphopeptide mapping of cAMP- and calmodulin-dependent phosphorylated protein, comigration with authentic synapsin I, and sensitivity to digestion with collagenase. Synapsin I and CaM kinase II were found in association with both microtubule preparations and preparations enriched in neurofilaments. Antibodies to synapsin I specifically labeled neurofilaments prepared in vitro. Immunocytochemical studies on rat brain tissue demonstrated synapsin I immunoreactivity specifically associated with the neuronal cytoskeleton as well as synaptic vesicles. The observed synapsin I staining on cytoskeletal elements was considerably diminished or abolished by the inclusion of Triton X-100 in the staining solutions. These results indicate that synapsin I is associated with the cytoskeleton and may be an important link between cytoskeletal elements as well as between the cytoskeleton and membrane.

Antibodies↗
PubMed1986

Reaction of Lewy bodies with antibodies to phosphorylated and non-phosphorylated neurofilaments.

The reaction of Lewy bodies in the substantia nigra and the sympathetic ganglia was studied in 10 cases of Parkinson's disease and other Lewy body-related conditions, using monoclonal antibodies to non-phosphorylated (02-40) and phosphorylated (03-44 and 07-5) neurofilaments. Although many Lewy bodies were unstained, a peripheral ring of positive immunoreactivity was frequently observed. This reaction was more common and more intense with antibodies to phosphorylated neurofilaments, suggesting a posttranslational phosphorylation of elements in the Lewy body. Although Lewy bodies appear to have some antigenic sites in common with neurofilaments, certain differences between neurofilaments and Lewy body filaments are emphasized.

Antibodies, Monoclonal↗
PubMed1986

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.

Aluminum↗
PubMed1986

Phosphorylated neurofilament antigens in neurofibrillary tangles in Alzheimer's disease.

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease (AD), and their presence correlates with the presence of dementia. A major constituent of NFT is the insoluble paired helical filament which shares some antigenic relationships with normal cytoskeletal elements, particularly neurofilaments. If neurofilament proteins (200, 145-160, and 68 kilodaltons [kd]) participate in the formation of NFT, the distribution of these constituents might be expected to be abnormal. To examine this issue, we used immunocytochemical methods to localize phosphorylated and nonphosphorylated epitopes of neurofilament proteins in hippocampal neurons of controls and patients with AD. Normally, the 200-kd neurofilament protein is not phosphorylated in the perikarya of neurons. However, in AD, many pyramidal neurons contained immunoreactive phosphorylated neurofilaments. Patterns of immunoreactivity (linear, flame-shaped, or skein-like within perikarya) greatly resembled the appearance of silver-stained NFT. This pattern of immunoreactivity was not present in hippocampal pyramidal neurons in controls, except in one aged patient in whom adjacent silver-stained sections revealed a few NFT. Patterns of immunoreactivity with antibodies for nonphosphorylated neurofilament proteins were similar in control and AD neurons. Our results indicate that some NFT are associated with abnormal distributions of high molecular weight phosphorylated neurofilament proteins. One domain of the 200-kd protein is believed to be a component of the side arms which link neurofilaments and interact with microtubules. Abnormal interactions of perikaryal neurofilaments could play a role in the genesis of NFT, and this abnormality of the cytoskeleton could contribute to the dysfunction of neurons at risk in AD.

Aged↗
PubMed1986

The unlabeled antibody method: comparison of peroxidase-antiperoxidase with avidin-biotin complex by a new method of quantification.

Upon plotting of areas against optical densities in immunocytochemically stained tissue sections, hyperbolic curves were obtained which could be reduced to two straight lines, one representing variations in stained structures, and the other variations in background. The slopes of the stained structure lines reflected staining intensity independently of total area of stained structure in a section. The ratio of slopes of the stained structure and background lines reflected immunocytochemical sensitivity. A comparison of the peroxidase-antiperoxidase (PAP) method with the avidin-biotin complex (ABC) method showed that at usual antibody dilutions the PAP method was much more sensitive than the ABC method, while at impractically high antibody dilutions it was moderately more sensitive. Once sufficient dilutions of antibodies were reached, staining intensities dropped sharply with the PAP method. On the other hand, the dilution curves were flat with the ABC method. The ABC method consequently appeared unsuitable for estimating variations in concentration of antigen or for distinguishing high or low concentrations of antigen. The ABC method provided a stain for myelin even in the absence of any antibodies.

Animals↗
PubMed1985

Aberrant neurofilament phosphorylation in Alzheimer disease.

Alzheimer tangles, despite their location in neuronal perikarya, react immunocytochemically with monoclonal antibodies to phosphorylated epitopes of neurofilaments. Normal perikarya do not contain phosphorylated neurofilaments. The aberrant phosphorylation in both plaques and tangles seems to be largely restricted to individual phosphorylation sites among the many sites available in neurofilaments. It is suggested that the Alzheimer lesion involves an imbalance within specific kinases responsible for phosphorylation of different sites in neurofilaments.

Alzheimer Disease↗
PubMed1984

An immunohistochemical study of myelin proteins during remyelination in the central nervous system.

The process of remyelination in the superior cerebellar peduncles of mice following demyelination with Cuprizone was studied immunohistochemically using antisera to myelin basic protein (MBP) and myelin-associated glycoprotein (MAG). Demyelination occurred after formation of myelinic vacuoles and resulted in almost complete loss of demonstrable MBP and MAG from the peduncle. Prior to the onset of remyelination, oligodendrocytes with cytoplasmic staining for both proteins appeared in the peduncle. These cells were then associated with remyelinating axons. The axons were remyelinated in clusters until the MBP and the MAG in the whole peduncle were reconstituted, although the axon sheaths were thinner than those in normal animals. The results show that the immunohistochemical distribution of MBP and MAG in remyelinating axons resembles that in normal axons, and that the expression of myelin proteins in oligodendrocytes during remyelination reverts to that seen during normal development.

Animals↗
PubMed1984

Myelin basic protein and myelin-associated glycoprotein in chronic, relapsing experimental allergic encephalomyelitis.

One-micron plastic sections of spinal cords from SJL/J mice with chronic relapsing experimental allergic encephalomyelitis (EAE) were reacted immunocytochemically with antiserum to myelin basic protein and myelin-associated glycoprotein. The distribution of myelin basic protein and myelin-associated glycoprotein in myelin sheaths was compared in acute and chronic areas of demyelination. No difference in the size of the lesion was seen with the two antisera. Myelin-associated glycoprotein was seen periaxonally in both normal myelin sheaths and sheaths which showed extensive splitting and ballooning as seen with toluidine blue stain and myelin basic protein antiserum. At least at the level of the light microscope, myelin basic protein antiserum gave intense staining of myelin while antiserum to myelin-associated glycoprotein showed little or no affinity to stain the myelin sheath itself, in contrast to other recent electron microscope observations. A few myelin basic protein or myelin-associated glycoprotein-containing oligodendrocytes were seen in lesion areas and remyelination by oligodendrocytes was rare. These observations are in agreement with findings from other models of EAE and multiple sclerosis where a primary loss of myelin has been implicated.

Animals↗
PubMed1984

The distribution of myelin-associated glycoprotein and myelin basic protein in actively demyelinating multiple sclerosis lesions.

Active plaques from 4 patients with multiple sclerosis were examined for myelin-associated glycoprotein (MAG) and myelin basic protein (MBP) using the peroxidase-antiperoxidase (PAP) immunocytochemical procedure applied to paraffin sections. MBP loss was intimately related to the presence of infiltrating macrophages which appeared to remove MBP-positive fragments directly off intact myelin sheaths. Phagocytosis of MAG-positive myelin sheaths was also observed. These findings support previous morphological studies that suggest that phagocytosis by macrophages of myelin attached to axons is an important mechanism of demyelination in multiple sclerosis.

Adult↗
PubMed1983

Immunocytochemistry of brain-reactive monoclonal antibodies in peripheral tissues.

Among a total of 135 tissue-reactive monoclonal antibodies previously prepared, 81 were brain-selective and were classified into neuronal and non-neuronal categories. The neuronal antibodies were again subdivided into antineurofibrillar, antiperikaryonal-neurofibrillar, and antisynapse-associated groups. On the basis of morphologic, developmental, biochemical, and pathologic criteria, the antibodies in at least two of these groups were found to detect heterogeneous antigens (called "neurotypes") rather than different antigenic determinants in single antigens. On examining the distribution in peripheral organs of staining patterns of 11 antineuronal brain-reactive antibodies, we now confirm that these antibodies are, indeed, largely brain-specific. In general, non-neuronal elements in liver, lung, heart, thymus, intestine, adrenal, and spleen remained unstained. However, most of the antibodies stained peripheral neural elements. Occasional antibodies did stain selected, non-neuronal structures. Four out of five antineurofibrillar antibodies stained nerve fibers in adrenal medulla, intestine and thymus. All of three antiperikaryonal-neurofibrillar antibodies also stained nerve fibers in the adrenal medulla, but not in other organs. Two out of three antisynapse-associated antibodies stained what appear to be nerve contacts on adrenal medullary cells, but not on any other peripheral cells examined. The non-neuronal peripheral staining patterns were restricted to selective nuclear staining exhibited by two out of five antineurofibrillar antibodies and the staining of macrophage and selected cardiac muscle nuclei by two of three antisynapse-associated antibodies. However, one antineurofibrillar antibody also stained the cytoplasm of selected liver cells. Among non-neuronally reacting antibodies, two antibodies stained nuclei of all cells except neurons in brain as well as peripheral organs. An antibody staining the ciliary epithelium of choroid plexus also stained basal bodies of ciliated bronchial epithelium. The overall data suggest that the specificity of brain-reactive antibodies is high and that their cross-reactivity with epitopes in non-nervous tissue is rare. In these cases, the antibodies seem to provide specific reagents for these additional structures as well as for their specific brain antigens.

Animals↗
PubMed1983

Why monoclonals?

Until recently, antibodies used in a variety of immunochemical techniques have been raised in laboratory animals by repeated injections of purified antigen and adjuvant. These antisera are a mixture of antibodies that may recognize unique or common antigenic sites and have different affinities. Such polyclonal antisera can vary considerably from animal to animal or even with different bleeds from one animal. The hybridoma technique of Kohler and Milstein (1975) permits the selection of a monoclonal antibody with a specific affinity that recognizes a specific antigenic determinant. Monoclonal antibodies to a variety of neural antigens have been produced by injection of "impure" antigen. Immunocytochemical findings with these monoclonal antibodies demonstrate some of the advantages and problems of hybridoma technology. Studies with these antibodies in normal and pathological (B,B'-iminodipropionitrile intoxication, Alzheimer's disease) conditions illustrate some of the potential applications of monoclonal antibodies.

Animals↗
PubMed1983

Microheterogeneity ("neurotypy") of neurofilament proteins.

Neurofilaments purified from adult rat brainstem by two methods were electrophoresed on NaDodSO4/polyacrylamide gels to separate the triplet proteins (approximate Mrs of 200,000, 155,000, and 68,000) which, in turn, were electroblotted onto nitrocellulose paper. On Coomassie blue-stained gels that were not electroblotted, the same banding pattern was seen with both methods of preparation. Immunocytochemical staining of the electroblots with each of five monoclonal antibodies revealed that three of the monoclonal antibodies were specific for the Mr 200,000 neurofilament protein and two, for both the Mrs 200,000 and 155,000 neurofilament proteins. None of the antibodies reacted with the Mr 68,000 band. The Mr 200,000 band could be resolved into doublet bands. Individual monoclonal antibodies reacted with either one or both of the Mr 200,000 doublets. The immunocytochemical staining of the neurofilament triplets on electroblots was compared to that of adult rat cerebellar paraffin sections. Each monoclonal antibody had a unique pattern of staining, reacting only with certain subpopulations of neurons or their processes. Correlation of the staining patterns in cerebellar tissue sections with those of neurofilament polypeptides on electroblots suggested that different neurofilament polypeptides can be localized to different structures and subpopulations of neurons and that molecular heterogeneity ("neurotypy") may be revealed within the Mrs 200,000 and 155,000 neurofilament polypeptides.

Animals↗
PubMed1983

Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ.

The immunocytochemical staining patterns of 37 neuron-specific monoclonal antibodies previously described fell into four groups: (i) anti-synapse-associated, (ii) anti-neurofibrillar, (iii) anti-perikaryonal-neurofibrillar, and (iv) a single antibody reactive with a widely distributed epitope that covered the patterns of groups ii and iii. Antibodies of groups ii, iii, and iv were shown to be specific to neurofilament triplet subunits, even though there was little overlap in staining patterns between groups ii and iii. We examined nine of these antibodies as to their ability to distinguish functional states of neurofilaments dependent upon phosphorylation. Upon digestion with phosphatase, electroblot staining of neurofilament components was abolished with the five antibodies from group ii, enhanced with the three antibodies from group iii, and unaffected with antibody iv. Immunocytochemical staining of Bouin-fixed paraffin sections of rat brain was unaffected by phosphatase pretreatment. With antibodies of group ii, digestion with trypsin also left staining unaffected, but when followed by digestion with phosphatase, staining was diminished with three out of five antibodies. In contrast, digestion with trypsin abolished all staining with each antibody from group iii. If followed by digestion with phosphatase, staining reappeared, but the group iii pattern was replaced by a group ii pattern. Staining of this pattern was again abolished upon a second treatment with trypsin. The antibody from group iv lost most of its groups ii and iii staining patterns when sections were digested with trypsin. The group ii pattern reappeared and, indeed, was enhanced upon a subsequent phosphatase treatment and was reduced again upon a second trypsin treatment. Staining by four out of five antibodies from group ii was inhibited by inorganic phosphate. The data indicate that certain nerve cell bodies, their dendrites, and at least proximal axons possess nonphosphorylated neurofilaments and that long fibers, including terminal axons, possess phosphorylated neurofilaments. We propose that phosphorylation may be a factor in stabilizing compacted forms of neurofilaments and that heterogeneity of the compacted structures may play a role in a possible multiplicity of function within individual nerve cells.

Animals↗
PubMed1982

Distribution of papovavirus, myelin-associated glycoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions.

To study how viruses interact with oligodendroglia and produce demyelination, we immunostained paraffin and epon sections of lesions from patients with progressive multifocal leukoencephalopathy (PML) with antisera to papovaviruses, oligodendroglial myelin-associated glycoprotein (MAG), and myelin basic protein (MBP) according to the peroxidase-antiperoxidase method. In paraffin sections from a rapidly progressive case of PML, hyperimmune JC virus antiserum stained single oligodendroglia which were located in white matter that appeared normal histologically and stained normally with MAG and MBP antisera. In zones surrounding areas of demyelination, virus containing oligodendroglia were most numerous and MAG staining of periaxonal regions was decreased, but there was little change in MBP staining. In demyelinated regions, both MAG and MBP staining were severely altered; also there was much less JC virus staining. In tissue from three other chronic cases, viral antiserum stained fewer oligodendrocytes and the differences in MAG and MBP staining were much less striking. In epon sections from two biopsies of central nervous system tissue, we studied the electron microscopic appearance of oligodendroglia that also had been stained by JC virus antiserum. Virions were present in all nuclei and in some cytoplasmic regions. The results suggest that changes in MAG distribution are useful indicators of early oligodendroglial abnormalities which can cause myelin breakdown.

Aged↗
PubMed1982

Developmental expression of neurotypy revealed by immunocytochemistry with monoclonal antibodies.

Adult and developing rat cerebella were stained immunocytochemically with six neuron-specific monoclonal antibodies obtained from spleen cells of BALB/c mice immunized with hypothalamus. Monoclonal peroxidase-antiperoxidase complex was used in the staining procedure. In the adult, three different staining patterns were seen with these antibodies. The general patterns have been designated as (1) neurofibrillar, (2) perikaryal-neurofribillar, and (3) synapse-associated. In addition, each antibody within a designated group showed a different immunocytochemical distribution. Some antibodies reacted widely, for example, with many neuronal perikarya and fibers, and their distribution increased with the development of the brain. Other antibodies had a more restricted distribution and stained only some structures within an area or pathway. To account for this type of restriction of distribution, we propose that there may be microheterogeneity of some of the antigens visualized and have called this microheterogeneity 'neurotypy'. A second type of restriction was also observed. With several antibodies a loss of staining occurred in the adult cerebellum in structures that had reacted during early development. These differences in staining probably reflect developmental regulation of the antigens (neurotypes).

Animals↗
PubMed1982

Neurotypy: regional individuality in rat brain detected by immunocytochemistry with monoclonal antibodies.

Hybridomas from spleen cell fusions of six BALB/c mice immunized with hypothalamus were analyzed by immunocytochemistry for antibodies reactive with paraffin sections of fixed rat brain. In a total of 135 antibody producers, 60% were brain specific. Among these, 54% reacted with glial elements, pituitary cells, or basal lamina of intracerebral capillaries, with little variation among individual hybridomas in each of these groups. Forty-six percent of brain-specific antibodies reacted with neuronal structures, localizing on nerve fibers, neurofibrils, or perikarya. Neuron-specific hybridomas could be classified into groups that localized in anatomically defineable overall patterns. Within these patterns individual hybridomas exhibited extensive qualitative localization diversity ("neurotypy"). Conceivably, the genetic message for a common "proantigen" within an overall pattern may be slightly modified during differentiation of a neuron, thus leading to minor variability in antigenic expression. During antibody formation, similar minor changes occur in the differentiation of the genetic message for the antibody variable region. Apparently, minor changes in the antibody combining site among groups of hybridomas is reflected in the detectability of minor neurotypic changes among differentiated neuronal proantigens. If neurotypy proves to be the result of single-base substitutions or of variability in alignment of peptide-coding exons, the Scharrer concept of the fundamental significance of neurosecretion could also become applicable to neuronal specialization.

Animals↗