Motor neuron-astrocyte interactions and levels of Cu,Zn superoxide dismutase in sporadic amyotrophic lateral sclerosis.

Copper, zinc superoxide dismutase (SOD1) is involved in neutralizing free radicals within cells, and mutant forms of the enzyme have recently been shown to occur in about 20% of familial cases of amyotrophic lateral sclerosis (ALS). To explore the mechanism of SOD1 involvement in ALS, we have analyzed SOD1 in sporadic ALS using activity assays and immunocyto-chemistry. Analyses of SOD1 activity in washed erythrocytes revealed no difference between 13 ALS cases and 4 controls. Spinal cord sections from 6 ALS cases, 1 primary lateral sclerosis (PLS) case, and 1 control case were stained using three different antibodies to SOD1. Since astrocytes are closely associated with motor neurons, antibodies to glial fibrillary acidic protein (GFAP) and vimentin were used as independent monitors of astrocytes. The principal findings from localizations are: (1) normal motor neurons do not have higher levels of SOD1 than other neurons, (2) there was no detectable difference in SOD1 levels in motor neurons of ALS cases and controls, (3) ALS spinal cord displayed a reduction or absence of SOD1-reactive astrocytes compared to the control and PLS cases, and (4) examination of GFAP-stained sections and morphometry showed that the normal close association between astrocytic processes and motor neuron somata was decreased in the ALS and PLS cases. These results indicate the disease mechanism in sporadic ALS may involve alterations in spinal cord astrocytes.

Reactive astrocytes are widespread in the cortical gray matter of amyotrophic lateral sclerosis.

The distribution of reactive astrocytes was examined in the cortical gray matter of non-motor and motor regions from cases of familial and sporadic amyotrophic lateral sclerosis (ALS) and compared to that of beta-amyloid deposits. By glial fibrillary acidic protein immunocytochemistry, patches of reactive astrocytes, characterized by multiple reactive astrocytes in a circular or patch-like formation, occurred in 12 of 15 ALS cases examined. These patches of reactive astrocytes were not restricted to the motor cortex but were found in the gray matter in ALS in all examined brain regions, including frontal, temporal, inferior parietal, cingulate, occipital, and motor cortices, from both familial and sporadic ALS cases. Reactive astrocytes were also found in the subpial region and at the gray/white matter junction. Because patches of astrocytes can occur in association with senile plaques, beta-amyloid was localized. By immunostaining, beta-amyloid deposits were observed in five of the 15 ALS cases: three cases had only early plaques, two had both early and classic plaques. The number of ALS cases with both astrocyte patches and amyloid plaques was four of 15, but typically astrocyte patches in ALS occurred without any evidence of an association with beta-amyloid deposits. Therefore, the astrocyte patches in ALS are not the result of beta-amyloid deposition. The widespread occurrence of reactive astrocytes, as patches in the cortical gray matter and in the subpial region and at the gray/white matter junction, is evidence of a widespread pathology in ALS cortex in both familial and sporadic forms of the disease.

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes.

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of the Torpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [3H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [3H]choline uptake in rat neocortical and striatal P2 preparations; and 3) [3H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [3H]choline uptake or [3H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.

Ventral ectoderm of Xenopus forms neural tissue, including hindbrain, in response to activin.

The peptide growth factor Activin A has been shown to induce complete axial structures in explanted blastula animal caps. However, it is not understood how much this response to activin depends upon early signals that prepattern the ectoderm. We have therefore asked what tissues can be induced in blastula animal caps by activin in the absence of early dorsal signals. Using whole-mount in situ hybridization, we compare the expression of three neural markers, N-CAM, En-2 and Krox-20 in activin-treated ectoderm from control and ventralized embryos. In response to activin, both normal and ventralized animal caps frequently form neural tissue (and express N-CAM) and express the hindbrain marker Krox-20. However, the more anterior marker, En-2, is expressed in only a small fraction of normal animal caps and rarely in ventralized animal caps; the frequency of expression does not increase with higher doses of activin. In all cases En-2 and Krox-20 are expressed in coherent patches or stripes in the induced caps. Although mesoderm is induced in both control and ventralized animal caps, notochord is found in response to activin at moderate frequency in control caps, but rarely in ventralized animal caps. These results support the idea that in the absence of other signals, activin treatment elicits hindbrain but not notochord or anterior neural tissue; and thus, the anterior and dorsal extent of tissues formed in response to activin depends on a prior prepatterning or previous inductions.

Reactive astrogliosis is widespread in the subcortical white matter of amyotrophic lateral sclerosis brain.

Widespread astrogliosis exists in the subcortical white matter in amyotrophic lateral sclerosis (ALS). As revealed by glial fibrillary acidic protein (GFAP) immunostaining, the gliosis has the morphological properties of an active process. It is present in the midfrontal, inferior parietal, temporal, cingulate, and occipital cortices, as well as in the motor cortex. Compared to matched regions from other neurological diseases, the gliosis in ALS does not appear to be the nonspecific result of a progressive, degenerative disease. In cell number and apparent cell size, the gliosis is comparable to that present in neurological diseases known to have white matter gliosis. Cytologically, the gliosis most closely resembles that present in cases of cerebral infarction. The basis for this similarity is unknown.

Major histocompatibility complex antigen expression in the affected tissues in amyotrophic lateral sclerosis.

Monoclonal antibody immunocytochemistry was used to examine spinal cord and muscle in amyotrophic lateral sclerosis for changes that would indicate ongoing or potential immune activity. Increased expression of class I and II major histocompatibility complex (MHC) antigens was seen in the affected areas of spinal cord. New MHC expression was concentrated in phagocytes, particularly in degenerating white matter in which they were dispersed in the tissue and also packed around blood vessels. MHC antigen was not revealed in motor neurons or skeletal muscle fibers. An anti-pan-T-cell monoclonal revealed small numbers of T cells in degenerating white matter. Similar changes have been seen in other neurodegenerative disorders. They suggest a potential for (secondary) cell-mediated activity in the affected areas rather than an ongoing MHC-restricted T-cell response. Vessel-associated phagocytes may be a source of antigen to peripheral lymphoid tissue, stimulating production of the autoantibodies that have been described.

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.

Monoclonal antibody Tor 23 binds a subset of neural cells in the human cortex and displays an altered binding distribution in Alzheimer's disease.

Recent evidence suggests that alterations in molecules of the external neuronal surface may be pivotal factors in Alzheimer's disease (AD) either as primary or secondary lesions. We are studying neuronal surface components with a library of monoclonal antibodies (MAbs) made to highly purified, exclusively cholinergic nerve terminals of the Torpedo ray. The most extensively characterized of the Tor MAbs. Tor 23, binds the external membrane of some human neuronal cells in culture. Our present findings demonstrate that Tor 23, in situ, binds the apparent limiting membrane of rare neurons of the human isocortex. Tor 23 binds, in addition, internally within a subpopulation of subcortical white matter astrocytes, as identified by colocalization with glial fibrillary acidic protein. Neuronal surface binding of Tor 23 parallels our findings in other species: astrocyte staining was not observed in other species and may be unique to human. Immunoblot analysis of white matter reveals one polypeptide band with a relative mobility of 115,000 +/- 15,000 daltons. In the mid-frontal cortex from cases of AD. Tor 23 immunopositive neurons are greatly reduced in number and immunopositive astrocytes are completely absent. The reduction of the neuronal surface epitope defined by Tor 23 supports the recent hypothesis that surface molecules are altered in AD. The absence of Tor 23 positive astrocytes opens an area for specific investigation: namely, the role subcortical astrocytes may play in the pathogenesis of AD.

An atlas of a rare neuronal surface antigen in the rat central nervous system.

The mature nervous system contains functional synaptic networks composed of neuronal sets and subsets whose identity and maintenance may rely on external surface molecules specific for these neuronal subdivisions. Such molecules may reside permanently on specific neurons, serving to identify those neurons within a complex population. From a collection of monoclonal antibodies made to the Torpedo cholinergic synaptosome preparation, we have identified several antibodies that bind the surface of some, but not all, neurons of the mature rat brain (Kushner and Stephenson, 1983; Kushner, 1984). In the present study we catalog which neurons of the entire rat brain and spinal cord are immunopositive for one of these antibodies, Tor 23. The atlas we have compiled can be used (1) on a practical level to guide affinity purifications and neuronal cell sorting, and (2) more esoterically to address whether surface antigenic sets of neurons share or define a common functional property. In the forebrain, Tor 23-positive neurons predominate as a rare cell type of the inner cortical laminae. In the midbrain, few cells stain. In the hindbrain, labeled neurons are dispersed among several nuclei. The exceptions to these observations, areas that almost exclusively contain Tor 23-immunopositive neurons, are the spinal cord ventral horn, the deep cerebellar nuclei, some cerebellar-related nuclei, selected auditory nuclei, the supraoptic and paraventricular nuclei of the hypothalamus, and the CA2 pyramidal neurons of the hippocampus. The neurons that bind Tor 23 are related biochemically by virtue of that shared epitope alone. Whether they are related in a functional manner is a separate issue. Tor 23 demarks many motor-related structures, specifically, the ventral horn motor lamina, the orofacial motor nuclei 5 and 7, the ambiguus nucleus, the deep cerebellar nuclei, the pontine reticulotegmental nucleus, the lateral reticular nucleus, the gigantocellular reticular nucleus, the red nucleus, and neurons within the motor and somatosensory cortices. The overlap of Tor 23 with motor structures suggests that Tor 23 is a motor system marker.

Torpedo monoclonal antibodies react with components of the human peripheral nervous system.

A panel of 141 monoclonal antibodies, generated to the Torpedo ray cholinergic nerve terminal preparation, were tested for binding to components of human nerve and muscle. Tested by immunofluorescence, 13 of the antibodies reacted with components of the human intercostal nerve, and 9 bound either muscle or blood vessels in a diversity of staining patterns. Results indicate that the antibodies identify a spectrum of different antigens. Some of the antibodies that cross react with the human nervous system have been studied in the ray and rat. In the human peripheral nerve and muscle, their cytochemical distribution is consistent with what has been observed in these other species. These antibodies, therefore, are likely to identify components of the neuromuscular junction that have been conserved in evolution from elasmobranchs to humans. The antibodies identified here are potentially useful tools for a molecular examination of human nerves and muscles in pathological conditions.

A human teratocarcinoma which expresses a rare neuronal cell surface antigen.

A molecular characterization of the events at the cell surface of neurons is pivotal for our understanding of how the nervous system is formed and maintained. This study of cell surface events in the human nervous system may be crucial to the study of human neurological maladies. NTERA-2 is a human teratocarcinoma which is unique amongst the teratocarcinomas for its ability to express many neurons. Tested for the binding of a monoclonal antibody Tor 23, which recognizes a surface antigen on rare and specific neurons, cultures of NTERA-2 cells contained cells with a neuronal morphology which bound the monoclonal antibody Tor 23 on their surface. The data indicate that Tor 23 antigen is a cell surface molecule with the same or very similar properties in rays, rats, and humans. The NTERA-2 cells are thus capable of expressing highly differentiated neuronal cell surface phenotypes and promise to be a powerful model system for the study of cell surface events of the human nervous system in vitro.

Monoclonal antibody Tor 23 recognizes a determinant of a presynaptic acetylcholinesterase.

A significant proportion of the acetylcholinesterase that is present in the electric organ of Torpedo californica exists as a presynaptic membrane molecule. The monoclonal antibody Tor 23 binds the Torpedo presynaptic nerve membrane where it recognizes a polypeptide of 68,000 daltons. Our present studies indicate that Tor 23 identifies acetylcholinesterase. From the homogenates of Torpedo nerve terminals, Tor 23 immunoprecipitates measurable esterase activity. Esterase precipitation was not observed with no Tor 23 added; nor was it observed with any other test antibodies, including other Tor antibodies, in particular, Tor 70, which binds, as does Tor 23, to the presynaptic nerve membrane. The esterase activity was specific for acetylcholinesterase. Our studies indicate the molecule defined by Tor 23 has the solubility properties described for that of presynaptic acetylcholinesterase: it is soluble in detergent-treated electroplax homogenates and insoluble in high-salt extractions. In sections of Torpedo back muscle, both nerve and endplate acetylcholinesterase can be detected histochemically. Tor 23 localizes to the nerve and is not clustered at the endplate. The utility of the antibody Tor 23 thus includes biochemical and histological analyses of the multiple forms of acetylcholinesterase.

A library of monoclonal antibodies to Torpedo cholinergic synaptosomes.

A library of monoclonal antibodies was generated to the cholinergic synaptosome. The immunogen was a preparation of highly purified synaptosomes from Torpedo electric organ. One hundred forty-one hybridoma cell lines were generated from the fusion of a single mouse. Tests reveal these cells produce antibodies with a vast range of neuronal specificities. The initial screen for specificity of antibody production was solid phase radioimmune binding to the original, highly purified synaptosome preparation. Subsequent tissue specificity tests have indicated that most antibodies are synaptosome-specific amongst the fish tissues tested: brain, liver, and purified synaptic vesicles. Less than 11% cross-react with liver. Many antibodies cross-react with frog and rat CNS. Localization within the frog and rat nervous tissue has revealed a vast array of antibody staining patterns. Some antibodies stain in a synaptic fashion. A few stain a restricted set of mammalian CNS neurons. Others define a broader set of CNS neurons. These Torpedo antibodies promise to be valuable probes with which to describe the molecular cell biology of the nervous system, of neurons in general, and of cholinergic neurons in particular.

A synaptic vesicle antigen is restricted to the junctional region of the presynaptic plasma membrane.

The plasma membrane of electric organ nerve terminals has two domains that can be distinguished by monoclonal antibodies. A library of 111 mouse monoclonal antibodies raised to nerve terminals from Torpedo californica contains 4 antibodies that bind specifically to the outside of intact synaptosomes. The distribution of the binding sites of these monoclonal antibodies on the outside of intact nerve terminals was examined by immunofluorescence and immunoelectron microscopy. The binding sites of 3 (tor23, 25, and 132) are distributed uniformly over nerve trunks and fine terminal branches. The binding site of the fourth (tor70) is restricted to synaptic junctional regions. This antibody, but not the other 3, recognizes a major component of synaptic vesicles, a proteoglycan associated with the inner surface of the vesicle membrane. The difference in the pattern of binding of these monoclonal antibodies suggests that the region of the plasma membrane containing active zones is antigenically distinguishable from other nerve terminal plasma membrane. We suggest that the antigen recognized by tor70 is externalized by exocytosis of synaptic vesicles while other plasma antigens take a different route to the surface. The unexpected observation that the vesicle antigen remains on the surface after exocytosis and is prevented from diffusion from the synaptic junctional region would be consistent with an interaction between the vesicle proteoglycan and elements of the synaptic cleft.

A neurochemical description of the dopaminergic innervation of the stomatogastric ganglion of the spiny lobster.

The spiny lobster stomatogastric ganglion has been shown to be innervated by catecholaminergic processes which derive from cells of large central ganglia (Kushner and Maynard, 1977). Biochemical evidence had indicated that the stomatogastric system synthesizes dopamine and not norepinephrine from tritiated tyrosine (Barker, Kushner, and Hooper, 1979). Studies reported here document that the stomatogastric ganglion itself contains dopamine, as measured with a sensitive endogenous assay. Moreover, the ganglion can synthesize dopamine from tritiated tyrosine or DOPA. Additionally, when incubated in tritiated dopamine, the ganglion takes up dopamine and protects it from degradation; this process is inhibited by cocaine. When incubated with 3H-tyrosine, small but measurable amounts of tritiated dopamine were detected in the medium surrounding the ganglion.

Location of interganglionic neurons in the stomatogastric system of the spiny lobster.

The stomatogastric ganglion produces distinct and complex patterned output driving the mastication and filtration of food. It does so with a small number of neurons whose properties and interconnections have been extensively examined. The motor patterns are subject to modulation and integration by neurons of other ganglia. This paper reports a search for interneurons of the four interconnecting ganglia of this system, using cobalt chloride backfilling techniques. It was determined that only a small number of neurons may interconnect these ganglia: (1) In the stomatogastric ganglion there are two to three small neurons and six large neurons with neurites projecting anteriorly towards the other ganglia. (2) In the two commissural ganglia there are one to three small neurons whose neurites project to the primary input nerve of the stomatogastric ganglion. (3) In the oesophageal ganglion there are three small neurons whose location would allow them to play a co-ordinating role in the output of all four ganglia.

Synthesis of dopamine and octopamine in the crustacean stomatogastric nervous system.

The spiny lobster stomatogastric nervous system synthesizes dopamine and octopamine in vitro from exogenous [3H]tyrosine. Each amine accumulates with a specific distribution among 9 separately analyzed regions within the system. Synthesis of other catecholamines was not observed. [3H]Dopamine is found in nerves, ganglia, and identified commissural ganglion cell bodies in which catecholamine histofluorescence has been demonstrated. The biosynthetic and histochemical data together indicate that dopaminergic cells send axons from the commissural ganglia to the stomatogastric ganglion neuropil along the same pathway followed by fibers that activate the pylroic motor network. The results support the hypothesis that dopamine mediates activation of the pyloric system in vivo, as observed in vitro. [3H]Octopamine accumulates primarily in the commissural and stomatogastric ganglia, where it may modulate neuronal activity, but octopaminergic cells and release sites within the stomatogastric system have not been identified.

Localization of monoamine fluorescence in the stomatogastric nervous system of lobsters.

Explorations into the crustacean stomatogastric nervous system have focused on the stomatogastric ganglion as a paradigm of neural mechanisms and integrative circuitry. Emerging evidence implicates dopamine as a fundamental modulator of the bursting pacemaker neurons and many of the intraganglionic rhythms. The present paper has employed formaldehyde-induced fluorescence in freeze-dried specimens, a microscopic histochemical technique which demonstrates anatomical structures containing particular putative transmitters, indoleamines and catecholamines. We have localized specific, formaldehyde-induced fluorescence in the lobster stomatogastric nervous system. This fluorescence occurs throughout the stomatogastric ganglion neuropil, in 4-8 cell bodies of the bilaterally paired commissural ganglia and in discrete and continuous neurites interconnecting these 3 ganglia. Variations of the basic fluorescence technique suggest that the specific fluorescence is catecholaminergic. Evidence from related biochemical studies supports this observation and indicates that the catecholaminergic fluorescence is dopaminergic since tests for norepinephrine are negative while concomitant tests for dopamine are positive.