Nestin-positive spheres derived from canine bone marrow stromal cells generate cells with early neuronal and glial phenotypic characteristics

Bone marrow stromal cells (BMSCs) isolated from humans and rodents have been shown to generate neural cells under specific culture conditions and after transplantation in the central nervous system. The apparent plasticity of BMSCs has therefore been a target of intensive research in attempt to develop a novel therapy for neurological diseases. Canines sustain neurological disorders (e.g., traumatic spinal cord injury) that closely mirror pathology of those in humans. Therefore, we evaluated neural differentiation properties of canine BMSCs to provide insights into basic characterization of these cells for future neurotransplantation trials in canine patients with neurological disorders. We demonstrate that canine BMSCs form spherical cellular aggregates on anti-adhesive culture substrate in serum-free culture media, which morphologically and phenotypically resemble spherical aggregates of neural progenitor cells, so-called neurospheres. Upon dissociation and subculture on adhesive substrate, canine BMSCs express neuronal (ss capital SHA, Cyrillic-tubulin) and glial (GFAP, A2B5, and CNPase) markers. Formation of spherical aggregates appears to be a critical preceding process for some of the glial marker expression (CNPase and A2B5). However, expression of more mature neuronal (MAP2) and glial (MBP) markers could not be induced with the protocol used in this study. We suggest that induction of canine BMSCs into cells with neural progenitor cell characteristics is possible and that these cells may have the potential for future cellular therapy for neurological disorders.     

Polypeptide composition of acetylcholine receptor purified from teleost and elasmobranch electroplax membranes

Acetylcholine receptor (AcChR) was solubilized and purified from membranes derived from electric organs of the marine fish Torpedo marmorata, Torpedo nobiliana, Narcine brasliensis, and of the freshwater eel, Electrophorus electricus, using techniques originally developed for Torpedo californica (27., 28.Biochem. Biophys. Res. Commun.49, 572–578; 1973, Biochemistry12, 852–856. The conditions used were identical in each case and the goal was to determine the degree of similarity between receptors from each source since conflicting reports have appeared with regard to polypeptide composition. The Torpedo and Narcine preparations were of high specific activity and exhibited four polypeptide components of apparent molecular weights 64, 59, 50, and 40 × 10³ upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Two components were observed upon gel electrophoresis in sodium cholate or upon sucrose density gradient centrifugation, representing monomeric and dimeric forms. Eel acetylcholine receptor exhibited three major subunits of apparent molecular weights 57, 49, and 40 × 10³. The amino acid and neutral sugar composition of the purified receptor preparations have been determined. The results support the contention that the receptor is composed of several types of polypeptide.     

Lipid composition and physical properties of membranes from C-6 glial cells with altered phospholipid polar headgroups

Growth of C-6 glial cells in media enriched in the polar headgroup precursors N,N-dimethylethanolamine, N-monomethylethanolamine or ethanolamine for 24 h resulted in the accumulation of the corresponding phospholipids to about 30% of total membrane phospholipid. Under these conditions the cholesterol to phospholipid ratios were unaffected. With the exception of arachidonic acid, which was significantly reduced in the lipids from cells grown in the presence of N-monomethylethanolamine, the fatty acid composition of cells grown under the various conditions was identical. The physical properties of membranes prepared from these cells were compared by electron spin resonance spectroscopy using spin-labelled stearic acid. Modifications in cellular phospholipid composition did not affect either the order parameter or the correlation time of fatty acid spin labels. Since there are no significant effects on the other membrane lipids and since the physical properties of the membranes are maintained, these modifications in phospholipid composition provide a valuable means for studying the role of phospholipid polar headgroups in the function of membrane-bound enzymes and hormone receptors in C-6 cells.     

Comparative study of GABA-T from glial cells, neuronal perikarya cells and synaptosomes

1. This paper presents a fast method of brain cell separation and a comparative study of GABA-T from different cellular compartments (glial cells, neuronal perikarya cells and synaptosomes). 2. The method of cellular separation offers the advantages of rapidity, ease and reproducibility. 3. The GABA-T from the three studied compartments had similar kinetic characteristics in respect of their Kms and Vmaxs. 4. The GABA-T needs PLP to reach its maximum activity; this dependence is related to the enzyme localization.     

Regulation of neuronal excitability by release of proteins from glial cells

Effects of glial cells on electrical isolation and shaping of synaptic transmission between neurons have been extensively studied. Here we present evidence that the release of proteins from astrocytes as well as microglia may regulate voltage-activated Na⁺ currents in neurons, thereby increasing excitability and speed of transmission in neurons kept at distance from each other by specialized glial cells. As a first example, we show that basic fibroblast growth factor and neurotrophin-3, which are released from astrocytes by exposure to thyroid hormone, influence each other to enhance Na⁺ current density in cultured hippocampal neurons. As a second example, we show that the presence of microglia in hippocampal cultures can upregulate Na⁺ current density. The effect can be boosted by lipopolysaccharides, bacterial membrane-derived stimulators of microglial activation. Comparable effects are induced by the exposure of neuron-enriched hippocampal cultures to tumour necrosis factor-α, which is released from stimulated microglia. Taken together, our findings suggest that release of proteins from various types of glial cells can alter neuronal excitability over a time course of several days. This explains changes in neuronal excitability occurring in states of thyroid hormone imbalance and possibly also in seizures triggered by infectious diseases.     

Polypeptide composition of cell membranes from chick embryo fibroblasts transformed by rous sarcoma virus.

Chick embryo fibroblasts were transformed by the Bryan high-titer strain of Rous sarcoma virus (RSV-BH), or a mutant (RSV-BH-Ta) inducing temperature-dependent transformation. Surface membranes from normal and transformed cells were isolated as membrane vesicles by differential centrifugation, and as cell ghosts after ZnCl2 treatment and separation in an aqueous two-phase system. These preparations were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate or phenol/urea/acetic acid. In general a greater resolution of individual bands was found in gels containing phenol/urea/acetic acid, which separates polypeptides on the bases of size and charge. Electrophoresis of preparations from nontransformed cells showed that two polypeptides (molecular weights 200 000 and 250 000) found in cell ghosts were missing in membrane vesicles. In cell ghosts, transformation by RSV-BH resulted in a significant decrease of the 250 000 molecular weight complex. Also a polypeptide (molecular weight 73 000) prominent in membrane vesicles from nontransformed cells was decreased in transformed cells. Surfaces from cells transformed by RSV-BH-Ta at 37 degrees C presented patterns similar to those for RSV-BH infected cells. Shifting these cells to 41 degrees C resulted in an increase in the 250 000 molecular weight complex, although the amount of this protein(s) never reached that found in noninfected cells. Inhibitors of RNA and protein synthesis failed to block the morphological changes occurring in RSV-BH-Ta cells after temperature shifts from 41 degrees C to 37 degrees C or vice-versa. The same inhibitors caused a reduction in the levels of the 250 000 molecular weight complex at both temperatures. These data indicate that these large membrane-associated polypeptides play little or no role in the morphological changes associated with transformation and its reversal.     

Polypeptide composition and spectral properties of light-harvesting chlorophyll ab-protein complexes from intact and trypsin-treated chloroplast thylakoid membranes

The polypeptide composition and spectral properties of isolated light-harvesting chlorophyll $\text{a}\text{b}\text{-}\text{protein}$ complexes from intact and trypsin-treated thylakoid membranes of Hordeum vulgare and Vicia faba are compared. The LHCP complexes consist of four distinct polypeptides with molecular weights between 21 000 and 25 000 occurring in equal relative amounts in the whole polypeptide spectra of thylakoid membranes. It is shown indirectly that the two major polypeptides very probably belong to different chlorophyll-proteins. The loss of a small segment from both polypeptides during trypsin digestion of thylakoids does not substantially alter the spectral properties and cation-mediated aggregation of isolated LHCP complexes.     

Base-exchange enzymic system for the synthesis of phospholipids in neuronal and glial cells and their subfractions: a possible marker for neuronal membranes

Abstract— The calcium-dependent incorporation of L-[3-¹⁴C]serine and [1,2^?14C]ethanolamine into the phospholipid of isolated neuronal and glial cells from rabbit brain was studied, and the distribution of the enzymic system among the correspondent subfractions was examined. The neuronal cell-enriched fraction was found to possess a much higher rate of exchange of both bases than the glial cell-enriched fraction. Among the sub-fractions isolated from the neuronal and glial cells, those corresponding to neuronal plasma membranes and microsomes showed a noticeably higher exchange of serine and ethanolamine compared to the corresponding subfractions from glia. Neuronal/glial ratios of about 6–8 were found for the exchange activity in both plasma membrane-enriched fraction and in microsomes. Synaptosomes and synaptosomal subfractions contained low activities. It is concluded that the calcium-dependent enzymic system for the exchange of serine, ethanolamine and other nitrogenous bases with endogenous phospholipid is concentrated mostly in the neuronal perikaryal membranes, and could be used as a neuronal marker.     

Phospholipid metabolism in neuronal and glial cells during aging

The incorporation of cytidine-containing precursors (CDP-Cho and CDP-Etn) into the main phospholipid classes of cellular fractions enriched in neurons and glial cells from whole rat brains of different ages was examined. The rate of synthesis of choline phosphoglycerides in neuronal homogenates significantly decreased with age up to 18 months; after this time no additional decrease was found. The decrease of CDP-Etn incorporation in neurons was found to be less significantly affected by age up to 18 months, but the enzymic activity decreased after 18 months of age. No changes were found in the corresponding glial activity at any age. Biochemical phenomena that occur in 18-month-old rat brain (aged animals) were compared with phenomena occurring in 2-month-old rat brain (adult animals). No significant variations of lipid composition were found in neurons from either 18-month-old or 2-month-old rat brain. These results, together with some kinetic parameters, suggest that ethanolamine and choline phosphotransferases are affected differently by aging.     

Presenilin 1 modifies lipid raft composition of neuronal membranes

Protein-lipid interactions in the nervous system may provide insight into the causes of neurological disorders. In this study, we elucidated if expression of human presenilin 1 (PS1) in a mouse model changes the physico-chemical properties of brain membranes. PS1 is a multifunctional transmembrane protein and part of the γ-secretase complex. This complex is critical for the production of the Alzheimer related amyloid beta peptide. Brain membranes isolated from mice expressing a human wild-type PS1 transgene are less fluid and contain higher cholesterol and sphingomyelin levels. Moreover, our data reveal significant changes in membrane micro-domains and indicate that PS1 induces the formation of lipid rafts.     

Heterogeneous distribution of enkephalin-degrading peptidases between neuronal and glial cells

Cultured neurones, astroblasts and astrocytes from murine brain have been screened with specific tests for the presence of peptidases capable of degrading enkephalin. Bestatin-sensitive aminopeptidases represent the major enkephalin-degrading activity in all cases. The dipeptidylaminopeptidasic activity is much higher in the neuronal than the glial cultures, whereas the opposite is true for the metallopeptidase called "enkephalinase". Only trace amounts of the dipeptidylcarboxypeptidase "angiotensin-converting enzyme" have been found. We conclude that bestatin-sensitive aminopeptidases on nerve cells are probable candidates for enkephalin-inactivating enzymes, whereas the "enkephalinase" on glial cells more likely serves a scavenger function.     

Polypeptide composition of chlorophyll-protein complexes from romaine lettuce

The protein moiety of the two major chlorophyll-protein complexes associated with chloroplast membranes of outer, dark green leaves of a romaine lettuce shoot (Lactuca sativa L. var. Romana) has been analyzed by discontinuous sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Complex II, also termed light-harvesting chlorophyll-protein complex, is shown to consist of a major polypeptide of 25 kilodaltons (kD) and two minor ones of 27.5 and 23 kD. The 25 kD subunit is the single largest polypeptide component of the chloroplast membranes, accounting for about 25% of their total protein. Complex I contains only high molecular weight subunits, the major one being at 67 kD, these subunits representing only a small percentage of the chloroplast membrane total protein.     

Cysteine sulfinic acid uptake in cultured neuronal and glial cells

The presence of an efficient high affinity uptake system for L-CSA has been demonstrated in cultured neuronal and glial cells of various types. In neurons and most glial cells L-CSA uptake is inhibited by acidic amino acids,L-glutamate andL-aspartate and requires sodium ions; however the sodium dependence varies from one cell type to the other. The characteristics of the uptake system change during cell maturation, especially in astroblasts. The predominance of CSA uptake in glial cells as compared to neurons, the similarity of the kinetic parameters and of the structural specficity ofL-glutamate uptake suggest that both excitatory amino acids are transported by a common system. In view of accumulating evidence, the present results are in agreement with a role of CSA as a neurotransmitter and as a precursor for taurine biosynthesis in the central nervous system.     

Microheterogeneity of tubulin proteins in neuronal and glial cells from the mouse brain in culture

The microheterogeneity of the alpha and beta isoforms of tubulin in brain cells in culture was studied. The cells were prepared from two precise regions of the embryonic mouse brain (ED15), the striatum and the mesencephalon. It was possible to maintain virtually pure cultures of neuronal or glial cells up to 1 and 4 weeks in vitro, respectively. The tubulin heterogeneity of striatal and mesencephalic neurons was found to be very similar after a few days in culture. More precise examination of pure neurons from the striatum revealed that their tubulin content after 7 days in vitro exhibited the same degree of complexity as a control extract from a 4 day-old mouse brain. In fact, we could detect the presence of at least six alpha and nine beta tubulin isoforms. Among these isoforms a specific family of beta proteins (beta' tubulin) and the more acidic alpha proteins were present. Since these isoforms have, up to now, been found only in tubulin extracts prepared from the nervous system, our experiments suggest that they belong to the neuronal subpopulation of this tissue. This point is reinforced by their complete absence from the tubulin proteins extracted from pure glial cells even after several weeks in vitro. These results lead us to propose that brain tubulin microheterogeneity is associated with the presence of neurons and not of glia and may, therefore, play a specific role in maintaining neuronal shape and function.     

Transcellular biosynthesis of cysteinyl leukotrienes in rat neuronal and glial cells

Leukotrienes are mediators of inflammation that belong to a family of lipids derived from arachidonic acid by the action of 5-lipoxygenase. Leukotrienes have been detected in the central nervous system in association with different pathological events, but little is known about their biosynthesis or function in the brain. When rat neurons and glial cells in primary culture were stimulated with the calcium ionophore, no significant biosynthesis of leukotrienes was detected using liquid chromatography/mass spectrometry (LC/MS) techniques. However, when exogenous LTA₄ was added to these cultured cells, both neurons and glia were able to synthesize LTC₄. Activated neutrophils are known to supply LTA₄ to other cells for transcellular biosynthesis of cysteinyl-leukotrienes. Since neutrophils can infiltrate brain tissue after stroke or traumatic brain injury, we examined whether neutrophils play a similar role in the central nervous system. When peripheral blood neutrophils were co-cultured with rat neurons, glia cells, and then stimulated with calcium ionophore, a robust production of LTC₄, LTD₄, and LTE₄ was observed, revealing that neurons and glia can participate in the transcellular mechanism of leukotriene biosynthesis. The formation of LTC₄ through this mechanism may be relevant in the genesis and progression of the inflammatory response as a result of brain injury.     

Protective peptides derived from novel glial proteins

In studying the mediators of VIP neurotrophism in the central nervous system, two glial proteins have been discovered. Both of these proteins contain short peptides that exhibit femtomolar potency in preventing neuronal cell death from a wide variety of neurotoxic substances. Extension of these peptides to models of oxidative stress or neurodegeneration in vivo have indicated significant efficacy in protection. These peptides, both as individual agents and in combination, have promise as possible protective agents in the treatment of human neurodegenerative disease and in pathologies involving oxidative stress.     

Ion dependent efflux from neuronal and glial cell cultures

A spontaneous efflux of choline originating from the cytoplasmic free choline compartment and, partly, from metabolized form was measured from neurons and glial cells in culture. The efflux was stimulated by an excess of K⁺ and by the absence of Ca²⁺ ions from the incubation medium in both types of culture. The two effects did not appear to be synergistic.

The stimulation produced by an excess of K⁺ (100 mM) was blocked in neurons by 0.5 μM BaCl₂ and in glia cells by 0.1 μM BaCl₂ (in the presence of 30 mM K⁺). The stimulation produced by the absence of Ca²⁺ instead was not blocked by Ba²⁺ ions in either of the two types of culture. The results suggest that the stimulation induced by K⁺ (high concentration and long time of incubation) might be of biochemical rather than physiological nature and that choline may be driven out of the cells in correlation with the K⁺ gradient. The greater sensitivity of glial cells to K⁺ ions may also suggest a supportive role of these cells with respect to neurons, as they seem capable of furnishing choline for neuronal needs during depolarization.