Muhammad Farooq

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

Abstract: The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white‐matter regions of rat brain, and in Vibratome sections as well as paraffin sections.

A MODIFIED PROCEDURE FOR ISOLATION OF ASTROCYTE‐ AND NEURON‐ENRICHED FRACTIONS FROM RAT BRAIN

Our previously published method for isolation of neurons with extensive processes (Farooqet al., 1977) has been modified to permit the isolation of both astrocyte‐ and neuron‐enriched fractions. Rat cerebral tissue is incubated with acetylated trypsin and disrupted. The cell suspension is separated first by differential centrifugation and then by gradient centrifugation on discontinuous Ficoll gradients. The method is reproducible and is applicable equally well to immature and adult animals. The yield of astrocytes of 57% particle purity, and higher weight purity, is 4–7 × 106 cells/brain, amounting to 1.5–2.0 mg of protein. The astrocytes appear to be a mixture of fibrous and protoplasmic types. The yield of neurons of 90% particle purity is 10–14 × 106 cells/brain, amounting to 2.4–3.0 mg of protein. A total yield of neurons of 28–37 × 106 cells/brain can be obtained at 70% purity. These preparations have been characterized by light microscopy and protein, RNA and DNA content.

Tumor necrosis factor-induced proliferation of astrocytes from mature brain is associated with down-regulation of glial fibrillary acidic protein mRNA

Previous results from this laboratory have shown that tumor necrosis factor (TNF) is mitogenic for bovine astrocytes in chemically defined (CO) medium. The maximum mitogenic response was detected with 200 U/ml at 48 h. We have now extended these studies to assess the effect of TNF on message levels for the intermediate filament proteins glial fibrillary acidic protein (GFAP) and vimentin. The results have shown that, whereas TNF had only a slight effect on vimentin mRNA, TNF induced a marked decrease to 4.3 ± 2.0% of controls in GFAP mRNA which was both time and dose dependent. The lowest effective dose was 50 U/ml and the maximal effective dose was 200 U/ml. Kinetic analysis of this response demonstrated that a marked decrease in GFAP mRNA was present at 12 h and continued to decrease through 72 h. To determine the reversibility of the TNF effect, astrocyte cultures were exposed to 200 U/ml TNF for varying periods of tee and then cultured in fresh CD medium. A 1‐h pulse with TNF was sufficient to reduce GFAP mRNA levels when measured 24 h later. However, cultures incubated with 200 U/ml TNF for 48 h followed by incubation in CD medium without TNF for 7 days showed that GFAP mRNA levels had returned to 60% of the control values. Nuclear runoff assays showed that the effect of TNF on GFAP mRNA was at the posttranscriptional level. Polyacrylamide gel electrophoretic analysis of astrocyte cytoskeletal proteins demonstrated that GFAP levels were reduced after a 5‐day incubation with 200 U/ml TNF whereas protein levels of vimentin and actin were not significantly changed. Western blots confirmed that GFAP levels were reduced to 36% of the control values. Thus the effect of TNF on GFAP mRNA expression was not due to a generalized effect on intermediate filament metabolism.

The Bulk Isolation of Oligodendroglia from Whole Rat Forebrain: A New Procedure Using Physiologic Media

Abstract: A method for the isolation of oligodendroglia from undissected rat forebrain is described. The method has been applied to brains from 10‐, 30‐ and 60‐day‐old rats. The procedure uses a balanced salt solution at pH 7.2 throughout. Tissue is briefly exposed to trypsin and DNase and dissociated and the cells are purified on a discontinuous sucrose gradient. The isolates were composed of 90% phase‐bright rounded cells having diameters after fixation of 7‐12 μm. The contamination was primarily by red blood cells and phase‐dark nuclei. Neurons and astroglia were lysed by the procedure. The method is reproducible and should be applicable to other ages of rat or to other species. The cells have been examined by light and electron microscopy and analyzed for protein and nucleic acids. None of the cell parameters measured, including total protein (58 pg/cell), varied significantly with age. With this new method it should be possible to carry out studies on the development and metabolism of oligodendroglia in small laboratory animals.

Carbonic anhydrase, 5'-nucleotidase, and 2',3'-cyclic nucleotide-3'-phosphodiesterase activities in oligodendrocytes, astrocytes, and neurons isolated from the brains of developing rats.

Abstract: The activities of three myelin‐associated enzymes, carbonic anhydrase, 5′‐nucleotidase, and 2′,3′‐cyclic nucleotide‐3′‐phosphodiesterase (CNP), were measured in oligodendrocytes, neurons, and astrocytes isolated from the brains of rats 10, 20, 60, and 120 days old. The carbonic anhydrase specific activity in oligodendrocytes was three‐ to fivefold higher than that in brain homogenates at each age, and, at all the ages, low activities of this enzyme were measured in neurons and astrocytes. The oligodendrocytes and astrocytes from the brains of rats at all ages had higher activities of the membrane‐bound enzyme 5′‐nucleotidase than was observed in neurons. In oligodendrocytes from 10‐ and 20‐day‐old rats, the 5′‐nucleotidase activity was two‐ to threefold the activity in the homogenates (i.e., relative specific activity = 2.0–3.0), and the relative specific activity of this enzyme in the oligodendrocytes declined to less than 1.0 at the later ages, concomitant with the accumulation of 5′‐nucleotidase in myelin. The CNP activity was always higher in oligodendrocytes than in neurons, but not appreciably different from that in astrocytes from 20 days of age onward. The relative specific activity of CNP was highest in the oligodendrocytes from 10‐day‐old rats but was lower, at all ages, than we had observed in bovine oligodendrocytes. These enzyme activities in oligodendroglia are quite different in amount and developmental pattern from those reported previously for myelin.

Ganglioside content of astroglia and neurons isolated from maturing rat brain: consideration of the source of astroglial gangliosides.

Previous biochemical and histochemical studies have failed to clarify the nature or quantity of gangliosides in CNS astrocytes. Using improved methodologies for bulk isolation of both neurons and astrocytes as well as for ganglioside purification, we find significantly higher ganglioside concentration in astrocytes and very similar thin-layer chromatography (TLC) patterns for the two cell types. However, in vivo labeling of glycoconjugates via intracerebral injection of [3H]glucosamine prior to cell isolation revealed a different picture: whereas glycoproteins were well-labeled in both cell types after labeling periods of 1-2 h, gangliosides were appreciably labeled only in neurons. With longer time periods (8-48 h) between injection and sacrifice, there was convergence of specific radioactivity of gangliosides from the two isolated cell preparations. These changes are compared to those observed in synaptosomes and microsomes that were isolated simultaneously. The results suggest limited ganglioside synthetic ability in astrocytes as compared to neurons, a conclusion supported by assay of UDP-galNAc:GM3 N-acetylgalactosaminyltransferase in the isolated cells. Nevertheless, the presence of ganglioside GM1 in a substantial portion of bulk-isolated astrocytes was demonstrated by indirect immunofluorescent detection of cholera toxin binding. Ideas on the reconciliation of these apparently contradictory phenomena, including the possibility of intercellular transfer and/or phagocytosis are discussed.

Glycerol Phosphate Dehydrogenase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase: Activities in Oligodendrocytes, Neurons, Astrocytes, and Myelin Isolated from Developing Rat Brains

Abstract: Glycerol phosphate dehydrogenase (GPDH), glucose‐6‐phosphate dehydrogenase (G6PDH), and lactate dehydrogenase (LDH) activities were determined in Oligodendrocytes, neurons, and astrocytes isolated from the brains of developing rats. The activity of each enzyme was significantly lower in both neurons and astrocytes than in Oligodendrocytes. The GPDH activity in Oligodendrocytes increased more than 4‐fold during development, and at 120 days cells of this type had 1.4‐fold the specific activity of forebrain homogenates. The G6PDH activities in Oligodendrocytes from 10‐day‐old rats were 1.4‐fold the activities in the forebrain homogenates. The activities of this enzyme in Oligodendrocytes were progressively lower at later ages, such that at 120 days the cells had 0.8 times the specific activities of homogenates. The Oligodendrocytes had 0.6 times the homogenate activities of LDH at 10 days, and this ratio had decreased to 0.2 by 120 days. These enzymes were also measured in myelin isolated from 20‐, 60‐, and 120‐day‐old rats. By 120 days the specific activities of G6PDH and LDH in myelin were <8% of the respective activities in homogenates. The GPDH activity in myelin was, however, at least 20% the specific activity in the homogenates, even in the oldest animals. It is proposed that LDH could be used as a marker for oligodendroglial cytoplasm in subfractions of myelin and in myelin‐related membrane vesicles.

Properties of Bovine Oligodendroglia Isolated by a New Procedure Using Physiologic Conditions

Abstract: A new class of procedures, previously shown to permit the isolation of pure oligodendroglia from whole rat cerebrum, has been applied with equal or greater success for the bulk isolation of this cell type from bovine white matter. Thus, the generality of this approach has been demonstrated. The bovine preparations have a purity of >90% intact, phase‐bright oligodendroglia and are obtained in a yield of 8 × 106 cells per gram of white matter. Within 1 day it is possible to obtain a preparation containing 60 mg of protein from a single cell type. These cells show a higher degree of ultrastructural preservation of all cytoplasmic constituents than previously obtained. The values for protein (33 pg/cell), DNA (5.4 pg/cell), and lipid (5–6 pg/cell) are very similar to those obtained with an earlier procedure. The cell lipids are rich in galactolipid, which comprises 20% of the total. The activity of the “myelin‐specific” enzyme, 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37), is 4.7 μmol/min/mg protein, similar to that obtained previously for isolated oligodendroglia and about 25–40% of that found in myelin. The activity of 5′‐nucleotidase (EC 3.1.3.5) in the cells is about 10% of that in myelin or white matter.

Gangliosides of cultured astroglia

Cultured astrocytes prepared from newborn rat brain and 13-day-old chick embryonic brain were analyzed qualitatively and quantitatively for ganglioside content. All preparations contained approximately the same total level: 2.4-3.4 micrograms N-acetylneuraminic acid (NeuAc)/mg protein. In contrast, the value for primary cultures of neurons from chick embryonic brain was 5.9. The non-hexosamine-containing species, GM3 and GD3, comprised 75-85% of the total in astroglial cultures, the remainder consisting mainly of structural types other than the gangliotetraose series; choleragenoid assay revealed the latter to be virtually absent or to comprise at most a few percent. Deficiency of gangliotetraose synthesizing ability was indicated by the very low level of UDP-GalNac:GM3 N-acetylgalactosaminyltransferase detected in the cells. Treatment of cultured astrocytes with astroglial growth factor 2 or dibutyryl cyclic AMP caused little if any change in quantity or pattern of gangliosides. The large majority of cells stained in a manner characteristic of astrocytes: positive for glial fibrillary acidic protein, negative for galactosyl ceramides. Staining with cholera toxin and anti-GM1 antibody was essentially negative, as was that with tetanus toxin, A2B5 monoclonal antibody, and antibody to GD3. All evidence thus points to cultured astrocytes of rat and chick brain containing appreciable gangliosides, most of which are GM3 and GD3 with the majority of the remainder comprising structures other than the gangliotetraose type.

The long term culture of bulk-isolated bovine oligodendroglia from adult brain

Oligodendroglia isolated from adult bovine brain by the method of Farooq et al. could be plated on polylysine-coated plastic dishes with an efficiency of 55-80%, and maintained in culture for as long as 4 months. The addition of cytosine arabinoside to the nutrient medium resulted in cultures that were approximately 90% oligodendroglia and 10% large fibroblasts. From 50 g of white matter 100-160 X 10(6) oligodendroglia, containing approximately 6-10 mg protein, could be obtained in culture. These small round cells started to send out processes at 5 days in vitro and by 2 weeks they formed an extensive network of processes. By immunofluorescence, all cells of this morphology were positive for galactocerebroside (GC) and myelin basic protein (MBP), and negative for glial filament protein and fibronectin. Most of the large flat cells were positive for fibronectin and negative for GC, MBP and glial filament protein. As the cultures aged the oligodendroglia tended to clump and blebs formed on the surface of both perikarya and processes. By 4 months they showed evidence of degeneration and detached from the substrate. Electron microscope examination showed that the cells had the appearance typical of oligodendroglia in situ. The somata were round to elliptical, with eccentrically placed nuclei, and were larger than freshly isolated cells. They grew directly on the substrate or on the surface of the fibroblasts. In older cultures the cells formed tight nests. The somata were enveloped by sheets of oligodendrocyte cytoplasm, sometimes having a myelin-like appearance. Gap junctions and small desmosomes were seen between oligodendroglial processes and between oligodendroglia and fibroblasts. The cytoplasm was characterized by a prominent Golgi apparatus, many mitochondria and lysosomes, scattered rough endoplasmic reticulum, free ribosomes, frequent centrioles and an abundance of microtubules. In cells from older cultures large vacuoles were common, and rarely they had multilamellar walls with alternating major and minor dense lines resembling myelin.