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.     

Amplification of prostaglandin E2 stimulation of luteinizing hormone-releasing hormone release from median eminence explants: a metal(II)-specific effect of chelated copper

We have previously demonstrated that extracellular copper amplifies prostaglandin (PG) E2 stimulation of the release of luteinizing hormone-releasing hormone (LH-RH) from explants of the median eminence area (MEA). Two questions were addressed: what is the active form of copper and the metal(II) specificity for copper action? MEA explants were incubated for 5 min in the presence of CuCl2 (ionic) or copper complexed to histidine (CuHis) at a concentration of 200 microM each and then for 15 min in the presence of 10 microM PGE2. It was found that chelated copper but not ionic amplified PGE2 action, and that the magnitude of PGE2 stimulation of LH-RH release was 3-4-fold in copper-treated than untreated tissue. Moreover, PGE2-stimulated release was directly related to the dose of CuHis. To test the metal specificity, MEA explants were incubated for 5 min with one of the following metal(II) complexes: CuHis, NiHis, FeHis, ZnHis, CdHis, MnHis, or BaHis (200 microM each) and then for 15 min with 10 microM PGE2. Controls were incubated with metal(II) complex or PGE2. Of these complexes, only CuHis and to a lesser extent NiHis stimulated LH-RH release. However, CuHis was the only complex that amplified PGE2 stimulation of LH-RH release. Thus, amplification is specific for copper. The finding that chelated copper but not ionic copper amplifies PGE2 is suggestive that the copper-interactive sites on the LH-RH neurons are not exposed to the extracellular space but that they are either embedded in the plasma membrane, facing the intracellular space, or in the cytoplasm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copper-induced release of immunoreactive α-melanotropin from isolated hypothalamic granules

Previously, we demonstrated that copper chelates stimulate the release of luteinizing hormone releasing hormone (LHRH) from isolated hypothalamic granules. To assess the generality of the copper-stimulated release process, we determined the effects of copper on the release of immunoreactive α-melanotropin (α-MSH₁) from isolated granules. When granules were incubated with various copper complexes, CuATP stimulated α-MSH₁ release by 54 ± 6% (mean ± S.E.), Cu tartarate by 56 ± 4%, CuBSA by 32 ± 5% and Cu histidine by 29 ± 2%. CuATP-stimulated α-MSH₁ release from granules incubated under N₂ was 57% of that incubated under air. Furthermore, the reducing agent dithiothreitol (DTT) inhibited CuATP-stimulated α-MSH₁ release (p < 0.01), whereas oxidized DTT did not do so. Pretreatment of granules with the thiol-blocking reagents iodoacetic acid or 5, 5'-dithiobis-(2-nitrobenzoic acid) inhibited CuATP-stimulated α-MSH₁ release by 52 ± 3 and 38 ± 4%, respectively. Thus, chelated copper, rather than ionic copper, is the active form of the metal and the action of copper involves the oxidation of thiols. These data are similar to those previously observed for the copper-stimulated release of LHRH. Hence, the effects of copper on the permeability of granule membranes may be a generalized phenomenon which underlies susceptibility of storage granules to the reduction-oxidation status of the cellular milieu.     

A reduction in the concentration of immunoreactive corticotropin, malanotropin and lipotropin in the brain of the aging rat

We have previously shown that the hypothalamic concentration of immunoreactive α-melanotropin (α-MSH_i) is markedly lower in the aging female rat than in the young rat. The current view is that α-MSH is derived from corticotropin (ACTH), and ACTH, in turn, is derived from a large molecular-weight precursor (pro-opiocortin); pro-opiocortin also serves as the precursor to β- and γ-lipotropin (LPH)^{10, 12, 22}. To ascertain if the age-related reduction in the concentration of α-MSH_i may be a result of a decline in the production of pro-opiocortin, we determined the content of immunoreactive ACTH (ACTH_i), α-MSH (α-MSH_i), γ-LPH (γ-LPH_i), and protein, in 3 regions of the brain of young (4 months) and old (26–28 months) female rats: the medial basal hypothalamus (MBH, the region containing the perikarya of the ACTH/MSH/LPH neurons^{9, 23, 28, 36}), the preoptic anterior hypothalamus (POA), and the thalamus (regions containing axons of these neurons). The concentration of ACTH_i, α-MSH_i (mol/mg protein), or γ-LPH_i (U/mg protein) in the MBH of old rats was 30–50% of that in the MBH of young rats. Moreover, the concentration of ACTH_i, α-MSH_i or γ-LPH_i in the POA and thalamus of old rats was also lower than that in the POA and thalamus of young rats. Based on these findings, we propose that aging causes a reduction in the production of pro-opiocortin in the brain of the female rat and that such a change may be related to the altered function of the brain of the aged.     

Human fetal brain cells in aggregate culture: A model system to study regulatory processes of the developing human neuropeptide Y (NPY)-producing neuron

Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the brain and it has been implicated in a wide range of brain functions, including mentation. The aim of this study was to establish a culture system of human fetal brain cells expressing NPY in a regulated manner. The NPY production in response to forskolin and phorbol 12-myristate 13-acetate (PMA) was taken as a criterion for regulated expression of NPY. Aggregates were formed from dissociated cells derived from the cerebral hemispheres of human fetuses (12.5–19 weeks' gestation) by constant rotation and were maintained in serum-free medium. A 24 hr exposure to 10 μM forskolin + 20 nM PMA led to a 2–6-fold increase in NPY content of the cultures, most of which (80–90%) was secreted into the medium. The latter consisted of two substances differing in size: one corresponding in size to proNPY and the other to NPY. Thus, forskolin + PMA led to an increased production of NPY. Exposure to PMA alone led to an increase in NPY production comparable to that seen after forskolin + PMA and this effect of PMA was dose-dependent. In contrast, forskolin alone did not induce NPY production. Conditioned medium, derived from monolayer cultures enriched with human astrocytes, enhanced NPY production in response to forskolin + PMA in an age-dependent manner. The NPY production by aggregates derived from a 12.5-week-, 14-week- and 18-week-old fetus was enhanced 3-3.6-fold, 1.6-2-fold and 1.1-fold, respectively. Thus, expression of the NPY neurons in this culture system is a regulated process. The NPY production is enhanced markedly by activation of the protein kinase C pathway and by an astrocyte-derived soluble substance(s). Based on these results, we propose that this culture system can serve as a model for the study of regulatory processes of the human developing NPY neuron.     

Mechanism of accelerated current decay caused by an episodic ataxia type-1-associated mutant in a potassium channel pore

In Kv1.1, single point mutants found below the channel activation gate at residue V408 are associated with human episodic ataxia type-1, and impair channel function by accelerating decay of outward current during periods of membrane depolarization and channel opening. This decay is usually attributed to C-type inactivation, but here we provide evidence that this is not the case. Using voltage-clamp fluorimetry in Xenopus oocytes, and single-channel patch clamp in mouse ltk- cells, of the homologous Shaker channel (with the equivalent mutation V478A), we have determined that the mutation may cause current decay through a local effect at the activation gate, by destabilizing channel opening. We demonstrate that the effect of the mutant is similar to that of trapped 4-aminopyridine in antagonizing channel opening, as the mutation and 10 mm 4-AP had similar, nonadditive effects on fluorescence recorded from the voltage-sensitive S4 helix. We propose a model where the Kv1.1 activation gate fails to enter a stabilized open conformation, from which the channel would normally C-type inactivate. Instead, the lower pore lining helix is able to enter an activated-not-open conformation during depolarization. These results provide an understanding of the molecular etiology underlying episodic ataxia type-1 due to V408A, as well as biophysical insights into the links between the potassium channel activation gate, the voltage sensor and the selectivity filter.     

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.     

Management of posttraumatic metadiaphyseal radioulnar synostosis

Posttraumatic radioulnar synostosis results in functional loss of forearm rotation. Treatment preference is to excise the synostosis when associated fractures have healed or when the process is radiographically static. Interposition material is used in the region of the proximal radioulnar joint or when the medullary canal of the radius or ulna is breached. Irradiation is limited to lesions at or proximal to the radial tuberosity. Postoperative management includes resting splint that holds the extremity in the extremes of forearm rotation, and intermittent active and passive range of motion exercises. Anti-inflammatory medications are used only during hospitalization. Results have shown a good functional arc of pronosupination, and no recurrence, especially when the process is limited to the midforearm.