F. Gremo

EXPRESSION OF BASIC FIBROBLAST GROWTH FACTOR AND ITS RECEPTORS IN HUMAN FETAL MICROGLIA CELLS

The presence of basic fibroblast growth factor (bFGF) and FGF receptors was investigated in microglia cells derived from human fetal brain long‐term cultures. Production of bFGF was suggested through the capability of microglial extracts to stimulate plasminogen activator (PA) synthesis in endothelial cells. The identity of PA‐stimulating activity with bFGF was confirmed by its high affinity for heparin and its cross‐reactivity with polyclonal antibodies to human recombinant bFGF. These antibodies recognized a cell‐associated Mr 18,000 protein as well as trace amounts of the Mr 24,000 bFGF isoform in Western blot. All microglial cells showed bFGF immunoreactivity in the cytoplasm and, sometimes, in the nucleus.

Acetylcholine and Dopamine Promote the Production of Platelet Activating Factor in Immature Cells of Chick Embryonic Retina

Abstract: We have previously shown that platelet‐activating factor (PAF), a naturally occurring lipid mediator of cell‐to‐cell communication, was produced by 3‐day‐old chick retina stimulated with acetylcholine (ACh) and dopamine (DA), but not with other neurotransmitters. ACh and DA stimulated PAF synthesis via a dithiothreitol (DTT)‐insensitive choline‐phosphotransferase, without affecting the acetyltransferase pathway, which was stimulated only by the calcium ionophore A23187. Therefore, we attempted to study the effects of neurotransmitters on PAF production and on the activities of the DTT‐insensitive cholinephosphotransferase and acetyltransferase in the developing chick embryo retina up to hatching. Our results show that PAF was produced already at 8 days of development, when retinal cells are still rather immature and ganglion and Mueller cells are the only differentiated cells. The stimulation of PAF production occurred with ACh and not with other neurotransmitters. In older stages, DA also stimulated PAF production, as already described in the chick after hatching. DTT‐insensitive cholinephosphotransferase and acetyltransferase activities were present in 8‐day‐old embryos, the earliest stage analyzed. Both enzymatic activities increased with age; DTT‐insensitive cholinephosphotransferase increased rapidly from day 12 up to day 18, whereas acetyltransferase activity increased linearly up to the time of hatching. To promote PAF production, ACh and DA activate DTT‐insensitive cholinephosphotransferase, but not acetyltransferase. The developmental sequence of PAF stimulation suggests that PAF production occurs through stimulation of ACh and D2‐dopamine receptors, which are mostly present in ganglion and amacrine cells. Synaptic maturation enhances the effects of ACh and DA. Our data taken together suggest that PAF may play a physiological role after the time of hatching, since light stimulation induces neurotransmitter release which can eventually lead to PAF production.

Role of fibroblast growth factor-2 in human brain: a focus on development

Trophic factors have gained a great degree of attention as regulators of neural cells proliferation and differentiation as well as of brain maturation. Very little is known, however, about their effects on human immature nervous system. In this paper, data on expression of fibroblast‐growth factor‐2 and its receptors are reviewed and discussed in the light of its possible role in human brain development.

Homogeneous longitudinal profiles and synchronous fluctuations of mitochondrial transmembrane potential.

This study reports for the first time (a) the longitudinal profile of the transmembrane potential (mΔψ) of single mitochondria using a Nernstian fluorescent probe and (b) the distribution of mΔψ fluctuations of mitochondria undergoing permanent depolarization. Our findings show that (1) mitochondria in different energetic conditions coexist in the same cell, (2) mΔψ is rather homogeneous along the entire length of single mitochondria, (3) mΔψ is not influenced by the surrounding cytoplasmic environment and (4) mΔψ fluctuations occur simultaneously in groups of mitochondria connected in a network. Taken together, these findings provide further evidence for a functional relationship between mitochondrial arrangement and energetic condition.

Expression of CD137 and its ligand in human neurons, astrocytes, and microglia: Modulation by FGF‐2

CD137 (ILA, 4‐1BB), a member of the tumor necrosis factor receptor family, and its ligand CD137‐L were assayed by RT‐PCR and immunocytochemistry in cultured human brain cells. Results demonstrated that both neurons and astrocytes expressed specific RNA for CD137 and its protein, which was found both on the plasma membrane and in the cytoplasm. Surprisingly, microglia, which also expressed CD137 mRNA, showed negative immunostaining. CD137‐L‐specific RNA was detected only in astrocytes and neurons. When brain cells were treated with fibroblast growth factor‐2 (FGF‐2), upregulation of CD137 but not of its ligand was observed in neurons and astrocytes. Protein localization was also affected. In microglia, an inhibition of RNA expression was induced by treatment, whereas CD137‐L remained negative. Our data are the first demonstration that human brain cells express a protein found thus far in activated immunocompetent cells and epithelia. Moreover, they suggest not only that CD137 and CD137‐L might play a role in interaction among human brain cells, but also that FGF‐2 might have an immunoregulatory function in brain, modulating interaction of the central nervous system with peripheral immunocompetent cells.

Differential expression of fibroblast growth factor receptors by human neurones, astrocytes and microglia.

EXPRESSION of fibroblast-growth factor receptors (FGFRs) was studied in human fetal neurones, astrocytes, and microglia in culture. Northern blot analysis showed that neurones, and microglia expressed the mRNAs for FGFR-1, FGFR-2, FGFR-3, FGFR-4 at different levels, whereas astrocytes expressed only FGFR-1, and FGFR-4 mRNAs. Immunocytochemical localization of FGFR-1 revealed that this receptor was predominantly localized on the axon hillock membrane in neurones, and was associated with the plasma membrane of ameboid, activated microglia, and of glial-fibrillar acidic protein positive astrocytes. The expression of various members of the FGFR family in all the cell types investigated implicates FGFs in human brain development, and functions.

Features and Functions of Human Microglia Cells

Microglia represent a population of resident cells of the central nervous system (CNS) which have attracted much interest because of their multiple functions. Two principal forms of microglia have been described. “Ameboid” cells, also referred as gitter cells or reactive microglia, which appear during the embryonal period and after brain lesions, are morphologically similar to macrophages (Giulian and Baker, 1986; Giulian, 1987). The second form of microglia, the “ramified” cells (Ling, 1981), appear during the late postnatal period and persist through adult life (Murabe and Sano, 1983). Although the nature of the relationship between ameboid and ramified microglia is controversial, ramified microglia are generally viewed as functional quiescent microglia that lack monocytic properties, but acquire ameboid features and reactivity in the presence of tissue damage (del Rio Hortega, 1919; 1932; Oemichen, 1983).

Human microglia cultures: A powerful model to study their origin and immunoreactive capacity

In this paper, we report that pure cultures of human microglia were obtained from long‐term astrocytic cultures of human fetal brain. After five to six months and repeated cell passages, macrophage‐like cells started to spontaneously form in vitro, so that in two to three weeks the whole culture was populated by them. These cells were grown up to over 50 passages in culture and analyzed for morphology, specific marker positivity, growth rate and major histocompatibility complex (MHC) antigen expression with or without gamma‐interferon (IFN) stimulation. We found that, regardless of embryonic age of original cultures (10–15 weeks of gestation), cultures showed a remarkable homogeneity and purity and over 90 stained for typical microglial markers. Under basal conditions, two cell subpopulations similar to those described in vivo, we observed: the reactive ‘ameboid’ type and the resting ‘ramified’ one, the latter increasing with time in vitro and cell passages. Both cell subpopulations were capable of active phagocytosis and of high‐rate proliferation. They spontaneously expressed low levels of MHC class II antigens, but were negative for MHC class I. Stimulation with gamma‐interferon lymphokine upregulated the MHC class II expression as well as the MHC class I heavy chain form in ameboid, ‘reactive’ cells but not in the ramified ones. We also found that β2 microglobulin, already expressed in basal conditions, was dissociated from HLA A‐B‐C molecules in lymphokine‐stimulated cells at early passages. The physiological significance of these data, as well as the possible correlation with in vivo ontogenetic modifications, are also discussed.

Developmentally regulated expression and localization of fibroblast growth factor receptors in the human muscle

Fibroblast growth factors (FGFs) are believed to play a key role in tissue differentiation and maturation. Thus, the expression of the four members of the high‐affinity tyrosine kinase FGF receptor family (FGFRs) and of the low‐affinity heparan sulphate proteoglycan binding sites, syndecan‐1 and perlecan, was studied in the human skeletal muscle during development. Northern blot analysis demonstrated a developmentally regulated expression of the mRNAs for FGFR‐1, FGFR‐3, FGFR‐4, whereas only traces of FGFR‐2 mRNA were found. Each receptor type had a different developmental pattern, suggesting an independent regulation. Signal for FGFR‐3 was retained only in the adult muscle. Among the low‐affinity FGF binding sites, perlecan was absent, whereas RNA transcript for syndecan‐1 peaked at week 13 of gestation, after which a significant decrease was observed.  Immunohistochemistry for FGFRs revealed that their localization changed with muscle maturation. At early embryonic stages, FGFR‐3 and FGFR‐4 had a scattered distribution in the tissue, and FGFR‐1 was found on myotube and myofiber plasma membranes. At later stages, FGFR‐1 positivity decreased and was found in a few areas of the muscle, FGFR‐3 was concentrated in the nuclei of some, but not all, muscle fibers, and FGFR‐4 maintained an association with plasma membrane. In adult tissue, weak positivity for FGFR‐3 and FGFR‐4 was observed in the connective tissue only.  When immunocytochemistry was performed on human fetal myoblasts in culture, confocal microscope analysis revealed a nonhomogeneous cell membrane distribution of FGFRs. Taken together, the data strongly suggest that developmentally regulated expression and cell distribution of FGFRs play a role during muscle maturation. Dev. Dyn. 1998;211:362–373.

Developmentally regulated expression and localization of dystrophin and utrophin in the human fetal brain.

Expression of dystrophin and the dystrophin-related protein utrophin has been studied in the human fetal brain both in vivo and in vitro. Results showed that both these proteins were developmentally regulated, even if their expression followed a different pattern. Utrophin was found since very early stages of development, reached a peak between week 15-20 of gestation, declining then, so that at week 32 was barely detectable. The protein was mainly found in neuronal cell bodies, partially associated to the plasma membrane, and in astrocytes cytoplasm. On the contrary, the brain form of dystrophin was first detectable at week 12, increased up to week 15 and then remained stable. Dystrophin localization was similar but not identical to utrophin. In neurons, it was also partially associated with the plasma membrane of cell body and axon hillock. However, the most was concentrated in the cytoplasm and in the processes, where it appeared associated to neurofilaments. Astrocytes were negative for brain dystrophin, but positive for the muscle isoform. Results suggest that utrophin and dystrophin are likely to play a key, though different, role in the immature brain. They help in understanding the basic mechanism(s) underlying cognition defects frequently observed in Duchenne and Becker dystrophic patients.