Nitric oxide (NO) plays an important role in the pathogenesis of neurodegenerative disease. It has been shown that neuronal NO synthase (nNOS), the enzyme that constitutively produces NO in brain, is a component of the dystrophin-associated protein complex. The absence of dystrophin causes Duchenne muscular dystrophy. Thus, we attempted to study whether or not a decrease of dystrophin expression would induce a modification in nNOS expression in cultured human neurons. Human fetal neuronal cultures were treated with antisense oligonucleotides against different isoforms of dystrophin and the expression of nNOS tested by RT-PCR and immunocytochemistry. Results showed that nNOS mRNA was significantly decreased by about 35% in neurons treated with brain-specific dystrophin (brain Dp427) antisense, whereas iNOS expression was not affected. Accordingly, a decrease in immunostaining for nNOS was observed in antisense treated neurons compared to controls. Expression of neuronal markers, such as bFGF or synaptophysin, was not affected by the same antisense treatment. Astrocytes were not affected by treatment, as shown by utrophin expression, a dystrophin-like protein that was not modified in pure astrocytic cultures. Thus, we conclude that a decrease of dystrophin in human neurons is associated with a decrease of nNOS expression.

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. © 2003 Wiley‐Liss, Inc.

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.

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.

Antisense oligodeoxynucleotides (ODNs) can potentially block the expression of a single gene in living cells by binding to a complementary messenger ribonucleic acid (mRNA). These molecules can enter cultured cells, recognize a specific gene sequence, and inhibit its expression. For this reason, antisense oligodeoxynucleotide strategies are largely used to understand specific gene functions in biological processes and during development. Several factors must be considered when treating cells with antisense and appropriate controls, which must be chosen to demonstrate that the effects of treatment are due to specific gene inhibition; there are many nonspecific effects that are not due to antisense mechanisms alone. This chapter describes the standard protocols for antisense treatment of human fetal brain cultures and for controls that are required to interpret results. An antisense molecule can be considered specific if it determines a significant decrease in target RNA and related protein levels and if there is no strong loss of cell viability. The interaction between antisense and RNA requires a minimum level of affinity.

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.

The subcellular heterogeneity of mitochondrial membrane potential (m Delta psi) was investigated in confluent and sub-confluent cultures of four cell types (human astrocytes, HEp-2, MDCK and Vero cells) in normal growth conditions, hypoxia and apoptosis, The distribution of high-polarized mitochondria, detected by the potential-sensitive probe JC-1, was found to depend on: (1) the proximity to the cell edge; (2) the local absence of cell-cell contacts; and (3) the local absence of acidic vesicles. Both hypoxia and apoptosis produced a general m Delta psi increase with different redistributions of high-polarized mitochondria, Hypoxic cells maintained high-polarized mitochondria for over 24 hours, until cells underwent necrosis, On the other hand, apoptotic cells showed an unexpected convergence of high-polarized mitochondria into an extremely packed mass at one side of the nucleus, in a stage preceding nuclear condensation, but correlated to the retraction of cell-cell contacts.

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. © 1998 Wiley‐Liss, Inc.

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).

Antisense oligonucleotides offer the potential to block the expression of specific molecules within the cell, thus providing a useful tool in cell function studies. In this paper, we tested the possibility to block dystrophin expression in in vitro cultured neurons with antisense oligonucleotides administration. Human fetal neuronal cultures were treated with different doses of antisense oligonucleotides against dystrophin, the protein coded by the Duchenne muscular dystrophy gene. Results showed that labelled oligonucleotides rapidly accumulated into cultured neurons, but were discarded 15–24 h after treatment. However, no effects could be observed until 3–4 days after treatment, when immunocytochemical staining for dystrophin was significantly decreased in treated neurones. This result was confirmed by polymerase chain reaction assay which showed a significantly lower expression of the dystrophin specific mRNA. Electron microscope observations confirmed that neurons were affected. Large inclusions or packed granules were detectable in their cytoplasm and in terminal endings. Neuronal nuclear membrane was sometimes shredded, so that nuclear shape was altered. These phenomena were dose-dependent, further substantiating the hypothesis of a specific effect of antisense treatment. This interpretation was supported by the absense of alterations when cultures were treated with mismatch or non specific antisenses. Since the function of dystrophin is still unknown, these data might help in understanding the role played by this protein in the developing brain.

Experiments were carried out to correlate the cytological localization of DNA polymerase alpha with the presence of its specific mRNA in human lymphocytes studied at different times after phytohaemagglutinin stimulation. Our data indicated that in resting cells it is not possible to detect DNA polymerase alpha protein or mRNA by Northern hybridization. By contrast, in stimulated cells the detection of mRNA specific for DNA polymerase alpha synthesis is possible after 16 h phytohaemagglutin stimulation, whereas immunolocalization is possible after only 4 h stimulation. Observation of cytological preparations from cells stimulated for times long enough to obtain mitoses surprisingly showed an intense immunoreaction in mitotic chromosomes treated with monoclonal antibodies to DNA polymerase alpha.

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 beta 2 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.