Brain Activation by Peptide Pro-Leu-Gly-NH2 (MIF-1)

MIF-1 (Pro-Leu-Gly-NH2) is a tripeptide for which the therapeutic potential in Parkinson's disease and depression has been indicated by many studies. However, the cellular mechanisms of action of MIF-1 are not yet clear. Here, we show the specific brain regions responsive to MIF-1 treatment by c-Fos mapping, and determine the kinetics of cellular signaling by western blotting of pERK, pSTAT3, and c-Fos in cultured neurons. The immunoreactivity of c-Fos was increased 4 hours after MIF-1 treatment in brain regions critically involved in the regulation of mood, anxiety, depression, and memory. The number of cells activated was greater after peripheral treatment (intravenous delivery) than after intracerebroventricular injection. In cultured SH-SY5Y neuronal cells, c-Fos was induced time- and dose-dependently. The activation of cellular c-Fos was preceded by a transient increase of mitogen-activated protein kinase pERK but a reduction of phosphorylated Signal Transducer and Activator of Transcription (pSTAT3) initially. We conclude that MIF-1 can modulate multiple cellular signals including pERK, and pSTAT3 to activate c-Fos. The cellular activation in specific brain regions illustrates the biochemical and neuroanatomical basis underlying the therapeutic effect of MIF-1 in Parkinson's disease and depression

CNS targets of adipokines

This is the author accepted manuscript. The final version is available from American Physiological Society via the DOI in this record.Our understanding of adipose tissue as an endocrine organ has been transformed over the last twenty years. During this time a number of adipocyte-derived factors or adipokines have been identified. This paper will review evidence for how adipokines acting via the central nervous system (CNS) regulate normal physiology and disease pathology. The reported CNS-mediated effects of adipokines are varied and include the regulation of energy homeostasis, autonomic nervous system activity, the reproductive axis, neurodevelopment, cardiovascular function, and cognition. Due to the wealth of information available and the diversity of their known functions, the archetypal adipokines leptin and adiponectin will be the focused on extensively. Other adipokines with established CNS actions will also be discussed. Due to the difficulties associated with studying CNS function on a molecular level in humans, the majority of our knowledge, and as such the studies described in this paper, comes from work in experimental animal models; however, where possible the relevant data from human studies are also highlighted

TNF stimulates nuclear export and secretion of IL-15 by acting on CRM1 and ARF6.

Interleukin (IL)-15 is a ubiquitously expressed cytokine that in the basal state is mainly localized intracellularly, including the nucleus. Unexpectedly, tumor necrosis factor-α (TNF) time-dependently induced nuclear export of IL-15Rα and IL15. This process was inhibited by leptomycine B (LMB), a specific inhibitor of nuclear export receptor chromosomal region maintenance 1 (CRM1). In the presence of TNF, LMB co-treatment led to accumulation of both IL-15Rα and IL-15 in the nucleus of HeLa cells, suggesting that CRM1 facilitates nuclear export and that TNF enhances CRM1 activity. Once in the cytoplasm, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested 4 h after TNF treatment. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and Golgi. This indicates different kinetics and possibly different trafficking routes of IL-15 from its specific receptor. The TNF-induced secretion of IL-15 was attenuated by pretreatment of cells by brefeldin A that inhibits ER-to-Golgi transport, or by use of domain negative ADP-ribosylation factor 6 (ARF6) that interferes with exocytotic sorting. We conclude that TNF abolishes nuclear localization of IL-15 and IL-15Rα by acting on CRM1, and it facilitates exocytosis of IL-15 with the involvement of ARF6

ARF6 localizes to plasma membrane and endosomes and it is crucial to IL-15 secretion after TNF treatment.

<p>HeLa cells were transfected with pCDNA3.1 (control), WT-ARF6, or DN-ARF6 plasmids 48 h before the assay. (A) In control cells and those overexpressing WT-ARF6, TNF treatment for 4 h increased IL-15 release. The cells overexpressing DN-ARF6 did not show a response. In none of the groups did PBS vehicle treatment increase IL-15 release. Data are representative of three independent experiments. *: p<0.05 compared with the control. (B) The overexpression of WT-ARF6 and/or TNF treatment seemed to increase the expression of IL-15 and IL-15Rα, shown by WB of whole cell lysates. (C) The potential interaction of ARF6 with IL-15 was shown by ICC. The left panel is ARF6 immunoreactivity (red, stained with anti-ARF6 in control cells and anti-HA in cells overexpressing ARF6) and the right panel is IL-15 immunoreactivity (green). In control cells, endogenous ARF6 showed a diffuse vesicular pattern of cytoplasmic distribution. In cells overexpressing WT-ARF6 that increased overall expression as well as cell surface distribution of ARF6, IL-15 signal was also increased and showed cell surface distribution co-localizing with WT-ARF6. In cells overexpressing DN-ARF6, the small increase of ARF6 signal remained cytoplasmic, and did not show co-localization with IL-15. Bar: 10 μm. D) Intracellular distribution of IL-15 Rα (green) did not show co-localization with endogenous ARF6 in control cells (stained with an ARF6 Ab, red) or in those overexpressing WT-ARF6 (middle panel) or DN-ARF6 (lower panel, HA Ab staining, red). Bar: 10 μm.</p

Intracellular trafficking of IL-15 and IL-15Rα.

<p>(A and B) Distribution of IL-15 and IL-15Rα in intracellular vesicles. After TNF treatment for 4 h, IL-15 showed partial co-localization with late endosomes but very little with other organelles tested. IL-15Rα showed co-localization with both early and late endosomes, and to a lesser extent with endoplasmic reticulum and the Golgi complex. (C) WB showing IL-15 levels in the cell lysate after TNF (5 ng/mL) or BFA (2 μM) treatment. (D) Cell viability after TNF or LMB treatment shown by ATP production. (E) ELISA of cell culture medium showing IL-15 level in response to TNF or BFA treatment. *: p<0.05 from the non-stimulated control.</p

Co-localization analysis.

<p>M1: fraction of green fluorescence (IL15 or IL15Rα) overlapping with red fluorescence (EEA or Rab7).</p><p>M2: fraction of red fluorescence (EEA or Rab7) overlapping with green fluorescence (IL15 or IL15Rα).</p

Role of astrocytic leptin receptor subtypes on leptin permeation across hCMEC/D3 human brain endothelial cells

Astrocytic leptin receptors (ObR) can be upregulated in conditions such as adult-onset obesity. To determine whether the levels and subtypes of astrocytic ObR modulate leptin transport, we co-cultured hCMEC/D3 human brain endothelial cells and C6 astrocytoma cells in the Transwell system, and tested leptin permeation from apical to basolateral chambers. In comparison with hCMEC alone, co-culture of C6 cells reduced the permeability of paracellular markers and leptin. Unexpectedly, ObRb overexpression in C6 cells increased leptin permeation whereas ObRa overexpression showed no effect when compared with the control group of pcDNA-transfected C6 cells. By contrast, the paracellular permeability to sodium fluorescein control was unchanged by overexpression of ObR subtypes. Leptin remained intact after crossing the monolayer as shown by HPLC and acid precipitation, and this was not affected by C6 cell co-culture or the overexpression of different ObR subtypes. Thus, increased expression of ObRb (and to a lesser extent ObRe) in C6 cells specifically increased the permeation of leptin across the hCMEC monolayer. Consistent with the finding that the most apparent regulatory changes of ObR during obesity and inflammation occur in astrocytes, the results indicate that astrocytes actively regulate leptin transport across the blood-brain barrier, a mechanism independent of reduction of paracellular permeability