Renato Rozental

Cytokine regulation of neuronal differentiation of hippocampal progenitor cells

THE signalling mechanisms governing haematolymphopoiesis and those regulating neural development may be closely related, as indicated by similarities of higher-order structure and function of the cytokines involved1, of the regional and temporal regulation of their transcription and translation2–6, and of their bioactivity7–10. Here we investigate this possible evolutionary connection using retroviral transduction of a temperature-sensitive mutant form of the SV40 large T antigen to develop conditionally immortalized murine embryonic hippocampal progenitor cell lines11–14. Treatment of these cells with cytokines that are thought to participate in progressive lymphoid maturation, immunoglobulin synthesis15–18 and erythropoiesis19,20 causes progressive neuronal differentiation, as defined by morphological criteria, successive expression of increasingly mature neurofilament proteins21–23, and the generation of inward currents and action potentials. The cytokine interleukin(IL)-11 induces expression of action potentials that are insensitive to tetrodotoxin, which is indicative of develop-mentally immature sodium channels24. By contrast, for expression of more mature action potentials24 (tetrodotoxin-sensitive) one of the interleukins IL-5, IL-7 or IL-9 must be applied in association with transforming growth factor-α after pretreatment with basic fibroblast growth factor. Our results suggest that the mechanisms regulating lineage commitment and cellular differentiation in the neural and haematopoietic systems are similar. Further, they define an in vitro model system that may facilitate molecular analysis of graded stages of mammalian neuronal differentiation.

Temporal expression of neuronal connexins during hippocampal ontogeny

Communication through gap junction channels provides a major signaling mechanism during early brain histogenesis, a developmental time during which neural progenitor cells are inexcitable and do not express ligand-gated channel responses to the major CNS neurotransmitters. Expression of different gap junction types during neurogenesis may therefore define intercellular pathways for transmission of developmentally relevant molecules. To better understand the molecular mechanism(s) by which growth and differentiation of neurons are modulated by gap junction channels, we have been examining the developmental effects of a specific set of cytokines on differentiation and gap junction expression in a conditionally immortalized mouse embryonic hippocampal neuronal progenitor cell line (MK31). When multipotent MK31 cells are in an uncommitted state, they uniformly express the neuroepithelial intermediate filament class VI marker, nestin, are strongly coupled by gap junctions composed of connexin43 (Cx43) and express connexin45 (Cx45) at the mRNA level. As these cells undergo neuronal lineage commitment and exit from cell cycle, they begin to express the early neurofilament marker, NF66, and coupling strength and expression of Cx43 begin to decline with concurrent expression of other connexin proteins, including Cx26, Cx33, Cx36, Cx40 and Cx45. Terminal neuronal differentiation is heralded by the expression of more advanced neurofilament proteins, increased morphologic maturation, the elaboration of inward currents and action potentials that possess mature physiological properties, and changing profiles of expression of connexin subtypes, including upregulation of Cx36 expression. These important developmental transitions are regulated by a complex network of cell cycle checkpoints. To begin to examine the precise roles of gap junction proteins in traversing these developmental checkpoints and in thus regulating neurogenesis, we have focused on individual members of two classes of genes involved in these seminal events: ID (inhibitor of differentiation)-1 and GAS (growth arrest-specific gene)5. When MK31 cells were maintained in an uncommitted state, levels of ID-1 mRNA were high and GAS5 transcripts were essentially undetectable. Application of cytokines that promote neuronal lineage commitment and cell cycle exit resulted in down-regulation of ID-1 and upregulation of GAS5 transcripts, whereas additional cytokine paradigms that promoted terminal neuronal differentiation resulted in the delayed down-regulation of GAS5 mRNA. Stable MK31 transfectants were generated for ID-1 and GAS5. In basal conditions, cellular proliferation was enhanced in the ID-1 transfectants and inhibited in the GAS5 transfectants when compared with control MK31 cells. When cytokine-mediated neurogenesis was examined in these transfected cell lines, constitutive expression of ID-1 inhibited and constitutive expression of GAS5 enhanced initial and terminal stages of neuronal differentiation, with evidence that terminal neuronal maturation in both transfectant lines was associated with decreased cellular viability, possibly due to the presence of conflicting cell cycle-associated developmental signals. These experimental reagents will prove to be valuable experimental tools to help define the functional interrelationships between changing profiles of connexin protein expression and cell cycle regulation during neuronal ontogeny in the mammalian brain. The present review summarizes the current state of research involving the temporal expression of such connexin types in differentiating hippocampal neurons and speculates on the possible role of these intercellular channels in the development and plasticity of the nervous system. In addition, we describe the functional properties and expression pattern of the newly discovered neuronal-specific gap junctional protein, Cx36, in the developing mouse fetal hippocampus and in the rat retina and brain.

Prospects for Rational Development of Pharmacological Gap Junction Channel Blockers

Connexin-null mice and human genetic gap junction diseases illustrate the important roles that gap junction channels play under normal conditions, and the neuro- and cardioprotective effects of gap junction blocking agents demonstrate that closure of these channels may be beneficial in certain pathological situations. This overview summarizes studies in which gap junction modifying reagents have been characterized, highlighting examples of agents for which selectivity for gap junction subtypes has been demonstrated. In addition, strategies for targeting connexin domains through peptide inhibitors are outlined, which may ultimately provide agents that are not only connexin-selective in their actions, but also affect only a subset of a gap junction channels gating responses.

Gap Junction-Mediated Bidirectional Signaling between Human Fetal Hippocampal Neurons and Astrocytes

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.

Nervous system diseases involving gap junctions

R. Rozental , A.C. Campos de Carvalho , D.C. Spray a,c Department of Neuroscience, Rose F. Kennedy Center, Albert Einstein College of Medicine, 1300 Morris Park AÕenue, Bronx, NY, 10461, USA Department of Internal Medicine, Vision Institute and IPTESP, Federal UniÕersity of Goias, Goiania, 74000, Brazil Institute of Biophysics ‘‘Carlos Chagas Filho’’, Federal UniÕersity of Rio de Janeiro, 21949, Brazil

Purification of cell populations from human fetal brain using flow cytometric techniques

We recently established primary cultures from dissociated second trimester human fetal brains using a novel spin seeding method and characterized cellular populations with distinct phenotypes in these cultures. Here, we report that these neural cultures can be dissociated to single-cell suspensions, sorted by size using flow cytometry and re-seeded to yield cultures selectively enriched for the neuronal and glial cell populations. Sorted neurons were highly homogeneous, viable and extended processes, by one day after re-seeding. These neurons expressed immunoreactivity for neurofilament protein, retained their GABAergic phenotype and were electrically excitable. Re-seeded astrocytes proliferated in culture and expressed glial fibrillary acidic protein. We describe the conditions required for the flow cytometric sorting and tissue culture assays as well as the morphological, immunocytochemical and electrophysiological characteristics of the sorted neuronal population.

Renal morphology in connexin43 knockout mice

Abstract Connexins (Cx) are a family of proteins that constitute the intercellular membrane channels of gap junctions. These junctions permit intercellular movement of ions and other molecules between cells, a property vital to organogenesis. Cx43 is a member of the family of channel-forming proteins that are essential for cell-cell communication of developmental signals. Studies demonstrate that Cx43 is observed in mesenchymal cells of 12-day gestation mouse kidney, a crucial period of renal development. In order to study the significance of Cx43 on renal developmental morphology, we evaluated the kidneys of embryos lacking the gene encoding for Cx43. Polymerase chain reaction (PCR) from tail specimens identified wild-type (WT), heterozygote (HT) and knock-out (KO) progeny. In situ RT-PCR displayed abundant Cx43 staining in glomeruli, vasculature, and tubules in kidneys obtained from WT progeny. In contrast, Cx43 expression was completely absent in kidneys isolated from the KO. Renal histology in all three groups displayed no significant differences. Renal size was similar and there was no evidence of dysplasia or cyst formation in the KO. Our results indicate that absence of Cx43, heretofore considered essential for renal development, does not affect early renal morphological development.

Gap junctions in the cardiovascular and immune systems

Gap junctions are clusters of intercellular channels directly connecting the cytoplasm of adjacent cells. These channels are formed by proteins named connexins and are present in all metazoan organisms where they serve diverse functions ranging from control of cell growth and differentiation to electric conduction in excitable tissues. In this overview we describe the presence of connexins in the cardiovascular and lympho-hematopoietic systems giving the reader a summary of the topics to be covered throughout this edition and a historical perspective of the discovery of gap junctions in the immune system.

CHAPTER 15 – Cell–Cell Communication: An Overview Emphasizing Gap Junctions

This chapter discusses the cell communication. Cells within the nervous system are responsive to blood–brain permeant hormones and nutrients and interact with other neural cells through long-range and local signaling mechanisms. Gap junction channels make up electrical synapses between neurons and form direct pathways for the diffusional exchange of metabolites and ions among glia. It synchronizes neuronal activity, provides pathways for second messenger and metabolite exchange, and may modulate cell growth, differentiation, and organization. Abnormal gap junction expression or function is associated with both genetic and somatic disease states, presumably contributing to the pathology through loss of the important intercellular pathway provided by these channels. As discussed in the chapter, in mice connexins are deleted by molecular genetic manipulation that provides model systems in which the roles of specific gap junction types can be explored in detail, and the contribution of functionally ablated gap junctions to different pathological situations can be directly assessed. Each gap junction channel is made of two mirror image symmetric components (one contributed by each cell) called “connexons” or “hemichannels,” and each connexon in turn comprises six homologous subunits, the connexin molecules.

Age dependence of tolerance to anoxia and changes in cytosolic calcium in rabbit renal proximal tubules

Calcium(Ca2+)-dependent processes mediate, in part, anoxic cell injury. These may account for the difference in sensitivity to anoxia between certain immature and mature renal cells. To address this question, we studied the effects of anoxia on cytosolic free Ca2+ concentration ([Ca2+]i), cell integrity, and transport functions in micro-dissected proximal convoluted tubules (PCT) of <3-week-old (newborn) and >12-week-old (adult) rabbits. Tubules were loaded with 10 ώM fura-2 AM by incubation for 60 min at 37° C,and then superfused with isosmotic saline solution gassed with either 95%O2-5%CO2) control group) or 95%N2-5%CO2 (anoxia group) for 30 min. [Ca2+]i was measured ratiometrically; cell damage was assessed by nuclear binding of propidium iodide (PI). Anoxia resulted in a fourfold increase in [Ca2+]i in adult tubules (from resting values of 245±10 to 975±100 nM, P<0.001), whereas in newborn tubules the rise was significantly less (from resting values of 137±5 to 165±5 nM, P<0.001 between anoxic groups). Transient exposure to 100 mM potassium chloride, which depolarizes the PCT cells, induced increases in [Ca2+]i from baseline, to 920±90 nM in tubules from adult and to 396±16 nM in those from newborn rabbits (P<0.001 between age groups). After exposure to ligands such as parathyroid hormone (PTH) and ATP, [Ca2+]i increased in both newborn and adult tubules, but to lower levels in newborn tubules. The response to PTH and ATP was transient in both age groups, [Ca2+]i returning to baseline levels after 2 min. Following anoxia, tubules from adult animals exhibited staining of all cell nuclei by 1 min exposure to PI, indicative of gross permeabilization of the cells. Nuclei of anoxic immatures tubules did not stain with PI. The sodium-depedent uptakes of a glucose analogue (14C-α-methyl-glucopyranoside) and phosphate (32Pi) were preserved in agarose-filled tubules of newborns after anoxia, whereas in those of adults recovery from anoxia was associated with drastic reduction in the uptake of these solutes. Overall, our results suggest that: (1) during anoxia, cell Ca2+ rises to critical levels in PCTs of adults compared with those of <3-week-old animals, (2) Ca2+ influx occurs via a pathway activated by exposure to high [K+]o, presumably voltage-sensitive Ca2+ channels or reversal of Na+-Ca2+ exchange, (3) these pathways are either less active or less abundant in proximal tubules of newborn compared with adult rabbits, and (4) secondary active transport activity and cellular integrity are well preserved after anoxia in PCT cells of newborn but not of adult rabbits.