Ayalla Barnea

Induction of functional and morphological expression of neuropeptide Y (NPY) in cortical cultures by brain-derived neurotrophic factor (BDNF): evidence for a requirement for extracellular-regulated kinase (ERK)-dependent and ERK-independent mechanisms.

Previous studies have demonstrated that brain-derived neurotrophic factor (BDNF) induces expression of neuropeptide Y (NPY) neurons in aggregate cultures derived from the fetal rat cortex. Using BDNF induction of NPY production and neurite extension of NPY neurons as functional and morphological criteria, respectively, we addressed the question: Does BDNF activate the extracellular-regulated kinase (ERK) pathway and if so, is activated (phosphorylated, P)-ERK required for the induction of both the functional and morphological expression of NPY? BDNF led to a rapid (30 min) and sustained (6 h) phosphorylation of ERK. PD98059 (PD, a specific inhibitor of the ERK kinase MEK), drastically inhibited, LY294002 (LY, a specific inhibitor of phosphatidylinositol-3-kinase, PI-3K) partially inhibited, and GF 109203X (GF, a specific inhibitor of protein kinase C) did not inhibit phosphorylation of ERK. A 24-h exposure to BDNF led to approximately 2-fold increase in the total culture content of NPY ( approximately 60% of which was secreted and approximately 40% remained in the aggregates) and to an abundance of neurite-bearing NPY neurons. BDNF-induced NPY produced and secreted into the medium was inhibited 73% by PD, 52% by LY and not at all by GF. In contrast, BDNF-induced NPY produced and sequestered in the aggregates was not inhibited by any of these inhibitors, suggesting a role for the ERK pathway in induced secretion of NPY. PD or LY did not inhibit BDNF-induced abundance of neurite-bearing NPY neurons. K252a (an inhibitor of TrkB-tyrosine kinase) abolished all the effects of BDNF assessed in our cultures. In summary, we demonstrate that TrkB-mediated activation of the ERK pathway is preferentially required for BDNF induction of NPY produced and secreted but not for the induction of the expression of neurite-bearing NPY neurons. Thus, BDNF induction of the functional and morphological expression of NPY is brought about by ERK-dependent and ERK-independent mechanisms.

Comparison of neurotrophin regulation of human and rat neuropeptide Y (NPY) neurons : induction of NPY production in aggregate cultures derived from rat but not from human fetal brains

Previous studies established that brain-derived neurotrophic factor (BDNF) induces neuropeptide Y (NPY) production and accumulation of NPY-mRNA in cultures of rat fetal brain tissues. In this study, we addressed the question: Are cultured human NPY neurons regulated by BDNF and/or by another member of the neurotrophin (NT) family of growth factors? Using aggregate cultures derived from human fetal cortical hemispheres, we assessed the effect of BDNF on NPY production varying the following experimental conditions: fetal and culture age; medium composition (with and without serum), dose and duration of exposure to BDNF, and neurotrophin species tested (BDNF, NT-4/5, NT-3 or NGF). Under none of these conditions did BDNF, NT-4/5, NT-3 or NGF induce an increase in NPY production. This was in contrast to forskolin + phorbol 12 myristate 13-acetate (PMA) which were highly effective in inducing NPY production, verifying that expression of NPY is a regulated process in these cultures. None of these neurotrophins enhanced the response to forskolin + PMA. By comparison, using aggregate cultures derived from rat fetal cortices, BDNF and NT-4/5 were equipotent in inducing NPY production but NT-3 and NGF were essentially ineffective. Moreover, the effects of BDNF or NT-4/5 and forskolin + PMA on NPY production were additive, indicating the involvement of distinct intracellular signalling pathways. Western blot analyses of human- and rat-derived aggregates indicated the presence of full-length Trk receptors which are tyrosine-phosphorylated in response to either BDNF, NT-4/5 or NT-3. Primary cultures of astrocytes (rat as well as human) were devoid of a functional TrkB receptor, strongly suggesting a neuronal expression of TrkB in the aggregates. Thus, a functional TrkB receptor is expressed by both the human and rat aggregates, but only the rat aggregates responded to BDNF or NT-4/5. These results are consistent with a difference in a post TrkB-receptor event(s) mediating BDNF action in the cultured human and rat fetal NPY neurons.

Evidence for regulated expression of neuropeptide Y gene by rat and human cultured astrocytes.

A series of studies from our laboratory have established that fetal rat and human neuropeptide Y (NPY) cortical neurons in aggregate cultures are differentially regulated. In a preliminary study we found that primary astrocytes produce substantial amounts of immunoreactive (IR) NPY. We addressed the question: Is astrocyte production of NPY-IR a regulated process? The effects of brain-derived neurotrophic factor (BDNF, 50 ng/ml), basic fibroblast growth factor (bFGF), substance P (1 microM), forskolin (10 microM), or phorbol 12-myristate-13-acetate (PMA, 20 nM) on NPY-IR production was tested on rat and human primary astrocyte cultures. Of these agents, PMA and bFGF markedly induced NPY-IR production by rat as well as human astrocytes, forskolin induced NPY-IR production by human but not rat astrocytes, and neither BDNF nor substance P induced NPY-IR production by rat or human astrocytes. The molecular size of PMA-induced NPY-IR was found to be consistent with that of proNPY. Moreover, PMA induced the accumulation of mRNA corresponding in size to the neuronal NPY-mRNA. Immunocytochemical analysis of human post-mortem neocortex revealed co-existence of NPY-IR with astrocyte markers. These results indicate that cultured astrocytes express NPY gene in a regulated manner and they support our proposition that in situ reactive astrocytes may express NPY gene under some physiological/pathological conditions.

A putative role for extracellular ATP: Facilitation of67copper uptake and of copper stimulation of the release of luteinizing hormone-releasing hormone from median eminence explants

We have previously shown that extracellular copper stimulates the release of the luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA), that chelated copper (Cu) but not ionic Cu is the active form of the metal, and that there is a direct correlation between the ligand specificity for 67Cu uptake and Cu action. In this study, we examined the possibility that extracellular ATP can serve as a ligand facilitating Cu action on the LH-RH neuron. Hypothalamic slices or MEA explants of adult male rats were used. It was found that ATP facilitates 67Cu uptake by hypothalamic slices when Cu:ATP molar ratio was 1:2000 but not 1:2. Keeping the [Cu] constant (150 microM) and varying [ATP], ATP facilitation of Cu stimulation of LH-RH release from MEA explants was found to be a saturable function of [ATP]; maximal facilitation occurred with 2.5 mM ATP. When the nucleotide phosphate specificity for facilitation of Cu action was assessed, ADP, ATP, alpha, beta-methylene-ATP (the non-hydrolyzable analogue of ATP) and GTP were equally effective, whereas AMP and adenosine were ineffective. These results indicate that extracellular ATP can facilitate Cu action on the LH-RH neuron and they are consistent with two mechanisms: (1) ATP facilitating Cu uptake and hence, Cu action and (2) ATP facilitating Cu action via an interaction with a purinergic receptor.

Interleukin-1β induces expression of neuropeptide Y in primary astrocyte cultures in a cytokine-specific manner: induction in human but not rat astrocytes

Previous studies have demonstrated that astrocyte cultures express neuropeptide Y (NPY) in a regulated manner, namely, phorbol ester leads to an increase in proNPY-mRNA and NPY production. In this respect, the behavior of astrocytes derived from the human fetal or rat neonatal brain is similar (Regul. Pept. 75 (1998) 293). Since astrocytes can be exposed to high levels of IL-1beta, we addressed the question: Does IL-1beta regulate NPY expression by the astrocytes? Primary astrocytes derived from the human fetal or rat neonatal cortex were cultured in serum-free medium. IL-1beta, but not IL-6 or TNF-alpha, led to an increase in NPY production dose-dependently. IL-1beta action manifested in the human but not in the rat astrocytes and it was completely abolished by IL-1 receptor antagonist. The responsiveness to IL-1beta did not diminish upon sub-culture of the astrocytes (five passages). In addition, IL-1beta led to an increase in the abundance of proNPY-mRNA, which was preceded by a rapid and transient increase in cFos-mRNA and a rapid and sustained increase in JunB-mRNA. In contrast to cFos/JunB, IL-1beta did not alter the abundance of cJun-mRNA. In summary, we demonstrate that IL-1beta induction of NPY expression in astrocytes is species- and cytokine-specific and that IL-1 receptor is involved. Moreover, induction of NPY expression is preceded by a rapid increase in the expression of two transcription factors (cFos, JunB) that have been previously (Oncogene 9 (1994) 2369; J. Neurochem. 70 (1998) 1887) implicated in transcriptional regulation of the human NPY gene.

REGULATED PRODUCTION AND SECRETION OF IMMUNOREACTIVE NEUROPEPTIDE Y BY AGGREGATING FETAL BRAIN CELLS IN CULTURES

The aim of this study was to establish a culture system of fetal brain cells that could serve as a model for the study of the developmental regulation of the neuropeptide Y (NPY) neuron. Single cell suspensions were prepared from the hypothalamic-olfactory tubercle region of 18-day-old rat fetuses, and aggregates were formed by incubation in serum-free medium under constant rotation. Aggregate formation was complete within 24-48 h, and cultures were maintained for up to 23 days. The content of immunoreactive (IR) NPY in the medium and in the aggregates increased progressively with time in culture and at each time point, the medium contained 5- to 10-fold more NPY-IR. A 48-hour exposure to forskolin resulted in a 2-fold increase in the accumulation of NPY-IR in the aggregates and in the medium, indicating that both production and secretion of NPY are regulated by the cAMP intracellular pathway. Sephadex gel filtration revealed the presence of proNPY- and NPY-size substances. The ratio of NPY- to proNPY-size substances increased progressively with age of the aggregates as well as in tissues obtained from perinatal rats of comparable age. Thus, production and secretion of NPY-IR in the cultured aggregates are regulated processes and hence, this culture system can serve as a model to study regulatory processes in the developing NPY neuron.

Further characterization of the process of in vitro uptake of radiolabeled copper by the rat brain

We have previously demonstrated that hypothalmic slices obtained from adult male rats accumulate 67Cu by two ligand-dependent, saturable processes: a high and low affinity process. To further establish the generality of these uptake processes, we defined the ligand requirements and the saturation kinetics of 67Cu uptake by tissue slices obtained from the newborn hypothalamus (HT); adult male hypothalamus, hippocampus, cortex, median eminence, and caudate nucleus; hypothalamus and hippocampus of castrated (14 days) males and of pregnant (19 days) and ovariectomized (14 days) females. It was found that ionic 67Cu2+ was poorly taken up by newborn HT and adult caudate, complexation with His enhanced 67Cu uptake 3-4-fold, and complexation with albumin inhibited 67Cu uptake. These ligand requirements are identical to those we have previously shown for the adult HT. When 67Cu uptake was evaluated under conditions optimal for the high or the low affinity process, for each process the dose response curves generated from these various tissues were very similar. In addition, we assessed the uptake of both components of the CuHis2 complex by incubating tissues with 67Cu3 H-His2 and found that the tissue ratio of 67Cu:3H was a sigmoidal function of the concentration of the Cu complex such that at greater than 5 microM, the ratio was about 3-fold greater than the medium ratio; indicating preferential uptake of 67Cu relative to 3H-His. The changes in isotope ratios were observed in newborn HT and adult HT, as well as caudate. These similarities in the ligand requirements and saturation kinetics of 67Cu uptake establish the generality of these two processes of in vitro uptake of copper in the rat brain.

Continuous exposure to brain-derived neurotrophic factor is required for persistent activation of TrkB receptor, the ERK signaling pathway, and the induction of neuropeptide Y production in cortical cultures.

We have previously demonstrated that brain-derived neurotrophic factor (BDNF) induces persistent neuropeptide Y (NPY) production in cortical cultures in an ERK1/2-dependent manner. In some studies, it was shown that BDNF leads to the downregulation of TrkB receptor and some of its downstream responses, whereas in others it does not. We examined whether the BDNF requirement for induction of persistent NPY production correlates with that for induction of phosphorylation of TrkB and ERK1/2. Continuous 24-h exposure to BDNF led to a 2- to 3-fold increase in NPY production (maximal level). While 1 h of BDNF exposure induced NPY production at a half maximal level, 8 h was required for induction of a maximal level. BDNF-induced NPY production was completely inhibited by co-exposure to TrkB-Fc fusion protein (TrkB extracellular domain fused to Fc) and partially inhibited by TrkB-Fc added 1 h after BDNF; TrkC-Fc did not do so. Activation of TrkB receptor was analyzed at two potential tyrosine phosphorylated sites, the activation loop and the Shc binding. BDNF led to coordinated phosphorylation of the two sites that persisted for 6-8 h, and this was not associated with changes in the content of TrkB protein. The presence of BDNF throughout the 6- to 8-h period was required for the persistent phosphorylation of TrkB and ERK1/2. Thus, continuous BDNF activation of TrkB is required for persistent activation of the ERK1/2 pathway and induction of NPY production. We propose that, within the time frame analyzed in this study, BDNF does not lead to the downregulation of TrkB receptor or of the biological responses leading to NPY production.

Female odors lead to rapid activation of mitogen-activated protein kinase (MAPK) in neurons of the vomeronasal system.

Pheromonal mediation of reproductive function proceeds along a neuroanatomical pathway that connects the vomeronasal organ (VNO) at the periphery with downstream target-sites in the amygdala and hypothalamus. The MAPK pathway is a prominent cascade linking receptor activation to induction of effectors such as c-Fos. We addressed the question: Does a specific pheromone stimulus lead to activation (phosphorylation, P) of MAPK in the VN system of the male mouse? Phosphorylation of MAPK in the VN system was evaluated 15-30 min and 1.5-2 h after exposure to female odors, using immunocytochemical techniques. A rapid and transient cytoplasmic expression of PMAPK was noted in the VNO with a unique distribution of the expressing neurons in columns extending over the entire basal to apical axis. A rapid and sustained expression was noted in most amygdaloid and hypothalamic VN target-sites and also in a few amygdaloid and hypothalamic sites outside the traditional VN system. The extent of expression and the subcellular compartmentalization (nucleus, cytoplasm, processes) of PMAPK were region-dependent. Of the VN target-sites, the accessory olfactory bulb (AOB) stood out in the lack of expression of PMAPK, in the high expression of the MAPK enzyme itself and in the massive of expression of c-Fos. This expression profile implicates another pathway(s) in mediating VNO signaling to the AOB. Our results are the first to demonstrate the use of PMAPK to trace functional pathways. Based on the wide cellular and intracellular expression of phosphorylated MAPK in the VN system, we propose that the MAPK pathway plays an important role in mediating female pheromone signaling in the male mouse.

An early and transient period of protein synthesis is required for induction of neuropeptide Y-mRNA by phorbol ester and forskolin in aggregate cultures of fetal brain cells

We have previously shown that both forskolin (F) and phorbol ester (P) induce neuropeptide Y (NPY) production by aggregate cultures formed from dissociated fetal rat brains. In this study, we addressed the question: Do F/P induce NPY-mRNA in aggregate cultures and if so, does induction require active protein kinases and on-going protein synthesis? On Northern blots, the NPY cDNA hybridized to a single species of about 0.8 kb. F and P each induced a time-dependent increase in NPY-mRNA relative abundance, and this was inhibited by staurosporine, an inhibitor of both protein kinase A and C. Cycloheximide (CHX) inhibited F/P induction of mRNA in a time-dependent manner. When aggregates were incubated with F+P for a total 12 h period, CHX added along with F+P (0 h) completely inhibited, CHX added 1.5 h after F+P partially inhibited, and CHX added 6 h after F+P did not inhibit the increase in NPY-mRNA. To rule out the possibility that this inhibitory profile reflects toxicity of CHX, blots were re-hybridized with a SRIF cRNA probe and, as expected for SRIF gene, a 12 h exposure to CHX did not inhibit F+P induction of SRIF-mRNA. Close inspection of the blots (derived from 1.5% agarose gels) suggested the presence in F/P-treated aggregates of 2 species NPY-mRNA [approximately 0.75 and approximately 0.85 kb, most likely differing in the length of poly(A) tail (Jamal et al., 1991)]; size-fractionation on a higher resolution gel (3% agarose) resolved the F/P induced mRNA into 2 distinct bands, while mRNA from untreated cultures migrated as a single band--the 0.75 kb.(ABSTRACT TRUNCATED AT 250 WORDS)