Neuron-specific expression of high-molecular-weight clathrin light chain.

High-molecular-weight forms of clathrin light chains LCa and LCb contain inserted sequences and are expressed in brain tissue but have not been observed in peripheral tissues. Monoclonal antibodies specific for the high-molecular-weight form of LCb and all forms of LCa were used to analyze their expression in different species and different neuronal cell types. High-molecular-weight light chains were found in bovine, rat, mouse, chicken, and human brain, indicating a conserved pattern of expression. Neuron-specific expression of the high-molecular-weight light chains was suggested by analysis of human brain gray matter and white matter. The former contained a higher proportion of light chains with insertion sequences. Immunohistochemical analysis localized the high-molecular-weight form of LCb to synapses and neuronal perikarya, but not to glial cells. Immunofluorescent labeling of cultured chicken dorsal root ganglia confirmed expression in neurons but not Schwann cells. These results indicate that the high-molecular-weight forms of clathrin light chains are restricted in expression and found in neuronal cells.

A clonal analysis of glial lineages in neonatal forebrain development in vitro.

Retrovirus-mediated gene transfer combined with triple immunostaining for astro- and oligodendroglial markers (antibodies to glial fibrillary acidic protein, GD3 ganglioside, and galactocerebroside, and the O4 antibody) was used to study clonal aspects of glial lineage in primary cultures of the neonatal rat striatum. We found two major clonal populations: astrocyte clones containing GFAP+, but GD3-, O4-, and GC- cells, and oligodendrocyte clones containing cells expressing various combinations of GD3, O4, and GC, with rare GFAP+ cells. These results indicate that astrocytes and oligodendrocytes belong to separate lineages in forebrain postnatal development.

Macrophage products IL-1 alpha, TNF alpha and bFGF may mediate multiple cytopathic effects in the developing eyes of GM-CSF transgenic mice.

GM-CSF transgenic mice develop eye disease during ontogeny that is mediated by autostimulated macrophages. The ocular pathology is characterized in part by corneal and vitreous neovascularization, pronounced GFAP expression by retinal Müller cells and degeneration of the retinal photoreceptor layer. The invading intraocular macrophages express the genes for the cytokines interleukin-1 alpha, tumor necrosis factor alpha and basic fibroblast growth factor, which may contribute to the multifaceted developmental ocular disorder. These cytokines, suspected to be angiogenic, may be responsible for neovascularization of the cornea in our transgenic animals. GFAP is normally made by astrocytes in the superficial retina and is induced in Müller cells in models of retinal degeneration. This protein is abnormally and copiously produced by Müller cells in the transgenic mice, which we suggest may be due to the release of cytokines from the invading macrophages. We suggest a mechanism by which autostimulated macrophages, through a perturbation of their normal developmental role, may be responsible for photoreceptor cell death in these transgenic animals.

Localization of the NGFI-A protein in the rat brain.

Antibodies are used to localize the NGFI-A protein in the rat brain. The protein is found in a wide variety of neurons. However, not all neurons are stained. The protein is either absent or present at undetectable levels in glial cells. Neuronal nuclei stain intensely, cytoplasmic staining is lighter. Seizures cause a detectable increase in the intensity of staining.

[The presence of aluminum in cerebral vessels in Alzheimer's disease]. / Uber das Vorkommen von Aluminium in Hirngefässen bei Alzheimer-Krankheit.

Morin staining is a specific method by which to detect aluminium in the brain. In cases of Alzheimer disease, aluminium was found to occur in neurons and glial cells, dense cores of senile plaques, primitive plaques, and intracortical congophilic vessels. Findings obtained are likely to suggest concomitant presence of aluminium and amyloid. Aluminium is assumed to have high affinity for amyloid. Aluminium is thus capable of overcoming the blood-brain barrier.

VIP modulation of cultured glial cells of the rat retina.

Cultured retinal glial cells from the rat are responsive to modulation by vasoactive intestinal peptide (VIP). VIP (1 X 10(-6) M) elevated the intracellular cyclic AMP concentration from the basal level of (4.4-11.1) p mole/mg protein to (354-440) p mole/mg protein in three minutes at 25 degrees C. The half-maximal concentration is 4.8 X 10(-8) M, which is similar to that observed in the cultured retinal glial cells from the chick embryo.

Subcellular localization of an intermediate filament protein and its mRNA in glial cells.

Eucaryotic mRNAs are generally localized in the cell body, where most protein synthesis occurs. We have found that mRNAs encoding the glial intermediate filament protein are spatially distributed in the glial cell cytoplasm close to the location of the glial filaments. Whereas the glial filament protein mRNA was located predominantly in the distal process, actin mRNA was found almost exclusively in the apical portion of the glial cell. This pattern of mRNA localization might provide a mechanism for synthesis of proteins in specific subcellular compartments by mRNA translation locally.

Histochemical study of the differentiation of microglial cells in the developing human cerebral hemispheres.

Applying nucleoside diphosphatase (NDPase) histochemistry, the appearance and differentiation of microglial cells in the developing human cerebral hemispheres were investigated by light and electron microscopy. In the pallium of the 38 days old human embryo, a few round NDPase-positive cells (round cells) were observed in the expanding zone. Although distinct blood vessels had not yet formed within the wall of the pallium, some cellular elements resembling haemopoietic cells were noticed in the expanding zone. In the 51 days old fetus, blood vessels displaying NDPase activity were seen in the mantle and marginal layers, and some invaded the matrix. Several round NDPase-positive cells were distributed, mainly around the vascular sprouts (primitive blood vessels) in the matrix. In the marginal layer, NDPase-positive cells exhibiting short cytoplasmic processes were encountered (poorly ramifying cells). In the 58, 66 and 82 days old fetuses, the round NDPase-positive cells were seen mainly in the matrix or subcortical layer where vascular sprouts were conspicuous and the poorly ramifying cells were in the subcortical and marginal layers. In the two latter fetuses, NDPase-positive cells showing long highly ramifying cytoplasmic processes (highly ramifying cells) were noted mainly in the marginal layer and sometimes in the subcortical layer. In the 5 months old fetuses, numerous NDPase-positive cells were distributed in the mantle, subcortical and marginal layers, and most of them appeared to belong to the populations of the poorly or highly ramifying cells. On the basis of the ultrastructural features, the round cells and highly ramifying cells were regarded as amoeboid cells and microglial cells, respectively. These findings suggest that at least some amoeboid cells are transformed into microglial cells via the stages of poorly ramifying microglial cells, and also that, in the human cerebral hemispheres, appearance of the microglial elements is closely related with vascularisation, especially in the early developmental stages.

Postnatal development of vimentin-immunoreactive radial glial cells in the primary visual cortex of the cat.

In kitten area 17 vimentin-like immunoreactivity is expressed in radial glial fibres up to one month postnatally, i.e. the time for which neuronal migration continues. During this time fibre density gradually decreases. A subpopulation of these fibres also contains S-100 protein and glial fibrillary acidic protein. However, these latter antigens disappear earlier than vimentin. In addition, vimentin immunoreactivity can be observed in astroglial cells of the white matter between the second and fifth postnatal week. Many of these cells resemble mature astrocytes but partially they have an intermediate morphology suggesting the possibility that they originated from radial glia. Such displaced radial glial cells' are also positive for S-100 protein both in the cortex and white matter. Thus it is conceivable that at least part of the astrocytes of mature cat visual cortex originate from vimentin- or S-100-immunoreactive radial glia.

Lineage, migration, and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker.

Previous studies described three different classes of glial cells in the developing CNS of the early Drosophila embryo that prefigure and ensheath the major CNS axon tracts. Among these are 6 longitudinal glial cells on each side of each segment that overlie the longitudinal axon tracts. Here we use transformant lines carrying a P element containing a 130 bp sequence from the fushi tarazu gene in front of the lacZ reporter gene to direct beta-galactosidase expression in the longitudinal glia. Using this molecular lineage marker, we show that 1 of the "neuroblasts" in each hemisegment is actually a glioblast, which divides once symmetrically, in contrast to the typical asymmetric neuroblast divisions, producing 2 glial cells, which migrate medially and divide to generate the 6 longitudinal glial cells. As with neuroblasts, mutations in Notch and other neurogenic genes lead to supernumerary glioblasts. The results indicate that the glioblast is similar to other neuroblasts; however, the positionally specified fate of this blast cell is to generate a specific lineage of glia rather than a specific family of neurons.

Ganglioside and lipid composition of bulk-isolated rat and bovine oligodendroglia.

We have examined the ganglioside composition of 30-day and 60-day postnatal rat oligodendroglia, adult bovine oligodendroglia, gray matter, white matter, and myelin and also the total lipid composition of the oligodendroglial preparations. The ganglioside patterns of rat and bovine oligodendroglia, as previously found for human oligodendroglia, were more complex than those of myelin. These data indicate that oligodendroglial perikarya can synthesize many brain type gangliosides, not all of which are incorporated into the compact myelin. Alternatively, the ganglioside composition of myelin may be altered in situ by the myelin-associated neuraminidase. In these two species, as in human, GM4 appears specific to oligodendroglia and myelin, while GD3 and GM3 are enriched in oligodendroglia but not myelin. In bovine oligodendrocytes GD3 is the major ganglioside. The total lipid concentration, as well as the percentage of cholesterol, sphingomyelin, phosphatidylinositol, and phosphatidylserine, differ for 30- and 60-day-old rat oligodendroglia and may be developmentally correlated with changes in myelin composition during myelinogenesis. There are also marked differences in the lipid composition of bovine oligodendroglia compared to rat oligodendroglia, with the former having more galactolipid and less ethanolamine phosphoglycerides.

Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer's disease beta-protein recognize amyloid and diverse glial and neuronal cell types in the central nervous system.

Studies were conducted to identify neural cells that synthesize and/or process cerebral amyloid using antisera and monoclonal antibodies (MAbs) raised to synthetic peptides based on the first 28 amino acids of the amyloid beta-protein. Using rabbit and mouse antisera, and 7 MAbs, sections of neocortex, hippocampus, cerebellum, and spinal cord from Alzheimer's disease (AD), Down's syndrome (DS), and control cases were probed. The antibodies produced 3 distinct immunohistochemical patterns: 1) staining restricted to neuritic plaque and blood vessel amyloid only (antisera, 1 of 7 MAbs); 2) immunoreactivity confined to cytoplasmic granules in diverse neuronal, glial (astrocytes, ependyma) and other (leptomeningeal, perivascular, choroid plexus) cells (1 of 7 MAbs); 3) a summation of these 2 patterns (5 of 7 MAbs). Controls resembled the AD and DS cases, except for a paucity of immunoreactive plaques and blood vessels in the controls. Immunoreactivity was reduced or removed by the peptides used to produce these antibodies. Formalin- and Bouins-fixed tissues reacted weakly or not at all with these antibodies while microwave denatured tissues reacted very intensely with them. Specific staining was enhanced by treatment of the tissue sections with Triton X-100, NaDodSO4, or trypsin. These studies significantly extend earlier studies that localized amyloid beta-protein precursor mRNA to human brain cells, and they suggest that the beta-protein, its precursor, and/or fragments thereof may exist in diverse neural cell types in AD, DS, and control brains.

Structural characterization of alpha-bungarotoxin-binding proteins from Aplysia californica.

Structural features of alpha-bungarotoxin-binding proteins from the marine mollusc Aplysia californica have been examined as a first step toward delineating their potential role in cholinergic neurotransmission. Protein blotting with 125I-alpha-bungarotoxin was used to identify binding proteins in membranes prepared from Aplysia muscle and nervous tissue. Binding proteins from both tissues exhibited similar physical characteristics, which distinguish them from the prototypical alpha-bungarotoxin-binding protein, the nicotinic acetylcholine receptor obtained from Torpedo californica electric organ membranes. Aplysia binding activities migrate with an apparent molecular weight of 250 kDa on sodium dodecyl sulfate (SDS) gels in the presence of reducing agents. Binding of alpha-bungarotoxin to blots of Aplysia membranes is abolished by exposure of samples to heat or to low pH but is unaffected by reduction-alkylation treatment. In contrast, the alpha-bungarotoxin-binding subunit of the acetylcholine receptor from Torpedo membranes migrates on SDS gels at 40 kDa. It retains binding activity following exposure to heat or to low pH, but binding is substantially diminished by reduction-alkylation treatments. Another distinguishing characteristic of the Aplysia binding activities is revealed by examining recovery of membrane alpha-bungarotoxin-binding on protein blots; the high recovery of Aplysia binding contrasts sharply with the low recovery of Torpedo binding activity. The high apparent molecular weight of the Aplysia alpha-bungarotoxin-binding activities, their most distinguishing feature, is similar to an alpha-bungarotoxin-binding activity recently identified in lower vertebrate brain. Covalent cross-linking with 125I-alpha-bungarotoxin demonstrates, however, that the mobility of both Aplysia binding activities is due to a multimeric protein that is unusually resistant to dissociation in SDS. The covalently radiolabeled Aplysia alpha-bungarotoxin-binding activity migrates at approximately 260 kDa on SDS gels when solubilized at room temperature. When it was boiled before electrophoresis, the mobility of the radiolabeled protein shifts to approximately 70 kDa. Resistance to dissociation in the absence of boiling may explain both the high recovery of activity on blots and the insensitivity to reductive alkylation. Conversely, dissociation of the multimeric complex upon boiling may explain the observed loss of binding activity. Our results demonstrate structural similarities and differences between Aplysia alpha-bungarotoxin-binding proteins and the Torpedo acetylcholine receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

A type II keratin is expressed in glial cells of the goldfish visual pathway.

The predominant intermediate filament proteins of the goldfish visual pathway consist of neuronal and non-neuronal isoelectric variants (58 kd). We have isolated a cDNA clone for the glial intermediate filament protein (ON3) from an optic nerve expression library. The predicted amino acid sequence of this clone reveals that it codes for a type II keratin representing the goldfish equivalent of mammalian keratin K8. K8 has been shown to be associated with embryogenesis and development. Unlike the mammalian visual system, the goldfish visual pathway displays a remarkable capacity for functional regeneration. The expression of K8, a protein not usually expressed in glial cells but shown to be associated with development, in the goldfish optic nerve may be involved with the processes of growth and regeneration in the goldfish visual pathway.

Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells.

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

Peripheral nerve lesion produces increased levels of major histocompatibility complex antigens in the central nervous system.

Proliferation of central nervous system (CNS) glia in response to peripheral nerve injury occurs without apparent participation of cells of the immune system. It is shown here that following transection of the rat facial nerve there is strongly elevated expression of class I, and to a lesser extent, class II antigens of the major histocompatibility complex (MHC) in the facial nucleus. It is demonstrated by double-immunofluorescence studies that the cells responsible for increased levels of MHC class I antigens are endogenous brain microglia. These findings emphasize the thought that microglia are immunocompetent cells, but, at the same time, raise the possibility for a non-immunological function of MHC antigens under conditions of neural regeneration.

Reactive glioma in intracranial sarcoma ('sarcoglioma'). Histology, electron microscopy and immunohistochemistry of two additional cases.

The clinicopathologic features of 2 cases of sarcoglioma are described. This rare type of mixed brain tumor is composed of a central core of sarcoma with peripheral distribution of gliomatous elements with gradual transition from reactive to neoplastic astrocytes. The immunohistochemical features showed a strict positivity to glial fibrillary acidic protein only in glial cells, a positivity to vimentin prevalently in sarcomatous cells, whereas the factor VIII-related antigen was negative in astrocytes as well as in sarcomatous cells. The different origin of two neoplastic components is demonstrated well by an ultrastructural study. The morphological observations together with an adequate clinical setting contribute to separate this specific entity from other mixed brain tumors.

[Immunocytochemical localization of cytochrome P-450scc in cultured rat oligodendrocytes]. / Localisation immunocytochimique du cytochrome P-450scc dans les oligodendrocytes de rat en culture.

Primary cultures of glial cells from newborn rat forebrain were tested after 3 to 4 weeks. Oligodendrocytes and astrocytes were characterized by immunofluorescence with monoclonal antibodies to galactocerebroside and glial fibrillary acidic protein, respectively. The cytoplasm of oligodendrocytes was specifically and intensely immunostained with monospecific polyclonal antibodies to the cytochrome P-450scc involved in the synthesis of pregnenolone from cholesterol. This observation brings additional support to the concept of "neurosteroids".

Boundaries defined by adhesion molecules during development of the cerebral cortex: the J1/tenascin glycoprotein in the mouse somatosensory cortical barrel field.

The distribution of the 200/220 KDa J1 glycoprotein (J1-200/220), within the developing vibrissae-related barrel field of the mouse somatosensory cortex, was studied by immunocytochemistry using a monoclonal antibody. J1-200/220, a member of the L2/HNK-1 family of adhesion molecules, also appears to be the mouse homologue of tenascin. J1/tenascin-positive barrel-like structures are visible in the somatosensory cortex between 24 and 48 hr after birth, with the molecule present in prospective barrel boundaries. Immunoelectronmicroscopy reveals labeling that is associated with glial and neuronal plasma membranes, as well as glial end-feet on blood vessels. A possible major source of J1/tenascin expression at this time is astrocyte precursor cells and radial glia. In the putative astrocyte precursor cells, immunolabeling was observed within organelles including the Golgi apparatus. At P6-7 J1/tenascin is most prevalent within prospective interbarrel septae. J1/tenascin-positive barrel boundaries are barely visible on P9 and not observed on P16. The findings indicate that J1/tenascin represents a major component of previously described "hidden" boundaries that we have seen during development using other methodologies. The expression of adhesion molecule-rich boundaries during the critical stages of barrel field formation indicates roles for such molecules during specific cerebral cortical pattern formation events.

Phase separation of myelin proteins in triton X-114: differential behavior of myelin basic protein in purified myelin and in cultured oligodendrocytes.

Rabbit central (CNS) and peripheral nervous system (PNS) myelin, as well as nonmyelinating pig oligodendrocytes in culture, were extracted at 0-4 degrees C with the nonionic detergent Triton X-114. The solubilized proteins were partitioned into the detergent-rich and detergent-depleted (aqueous) phases that form upon heating to 37 degrees C. The proteolipid protein (PLP), myelin-associated glycoprotein (MAG), myelin oligodendrocyte glycoprotein (MOG) and P0 extracted from myelin were found exclusively in the detergent phase which is characteristic of the intrinsic membrane proteins. This was also the case for Wolfgram protein (WP), although this protein lacks transmembrane domains. A small fraction of the MAG and MOG extracted from oligodendrocytes partitioned into the aqueous phase, suggesting an altered conformation outside myelin or a different state of glycosylation. P2 and myelin basic protein (MBP) showed distinct patterns of behavior. P2 was found mainly in the aqueous phase giving strong support to its theoretically predicted conformation. Eighty-nine percent of the MBP extracted from CNS myelin and 81% of the pure MBP partitioned into the detergent phase. Surprisingly, most of the MBP extracted from the oligodendrocytes was recovered in the aqueous phase. We speculate that, in these cells, a hydrophilic protein might bind to the MBP in a specific manner, thereby preventing it from binding inappropriately to cellular components before its insertion into myelin.