Paracrine signaling by glial cell-derived triiodothyronine activates neuronal gene expression in the rodent brain and human cells

Hypothyroidism in humans is characterized by severe neurological consequences that are often irreversible, highlighting the critical role of thyroid hormone (TH) in the brain. Despite this, not much is known about the signaling pathways that control TH action in the brain. What is known is that the prohormone thyroxine (T4) is converted to the active hormone triiodothyronine (T3) by type 2 deiodinase (D2) and that this occurs in astrocytes, while TH receptors and type 3 deiodinase (D3), which inactivates T3, are found in adjacent neurons. Here, we modeled TH action in the brain using an in vitro coculture system of D2-expressing H4 human glioma cells and D3-expressing SK-N-AS human neuroblastoma cells. We found that glial cell D2 activity resulted in increased T3 production, which acted in a paracrine fashion to induce T3-responsive genes, including ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), in the cocultured neurons. D3 activity in the neurons modulated these effects. Furthermore, this paracrine pathway was regulated by signals such as hypoxia, hedgehog signaling, and LPS-induced inflammation, as evidenced both in the in vitro coculture system and in in vivo rat models of brain ischemia and mouse models of inflammation. This study therefore presents what we believe to be the first direct evidence for a paracrine loop linking glial D2 activity to TH receptors in neurons, thereby identifying deiodinases as potential control points for the regulation of TH signaling in the brain during health and disease.NIHFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Hungarian Scientific Research FundHungarian Academy of SciencesUniv Miami, Miller Sch Med, Div Endocrinol Diabet & Metab, Miami, FL 33136 USAUniversidade Federal de São Paulo, Mol Endocrinol Lab, Div Endocrinol, Dept Med, São Paulo, BrazilHungarian Acad Sci, Inst Expt Med, Lab Endocrine Neurobiol, Budapest, HungaryBrigham & Womens Hosp, Thyroid Sect, Div Endocrinol Diabet & Hypertens, Boston, MA 02115 USATufts Med Ctr, Div Endocrinol Diabet & Metab, Dept Med, Tupper Res Inst, Boston, MA USATufts Univ, Sch Med, Dept Neurosci, Boston, MA 02111 USAUniversidade Federal de São Paulo, Mol Endocrinol Lab, Div Endocrinol, Dept Med, São Paulo, BrazilNIH: DK77086NIH: DK37021FAPESP: 05/55825-8FAPESP: 05/55826-4Hungarian Scientific Research Fund: OTKA K81226Web of Scienc

Molecular Interpretation of ACTH-β-Endorphin Coaggregation: Relevance to Secretory Granule Biogenesis

Peptide/protein hormones could be stored as non-toxic amyloid-like structures in pituitary secretory granules. ACTH and β-endorphin are two of the important peptide hormones that get co-stored in the pituitary secretory granules. Here, we study molecular interactions between ACTH and β-endorphin and their colocalization in the form of amyloid aggregates. Although ACTH is known to be a part of ACTH-β-endorphin aggregate, ACTH alone cannot aggregate into amyloid under various plausible conditions. Using all atom molecular dynamics simulation we investigate the early molecular interaction events in the ACTH-β-endorphin system, β-endorphin-only system and ACTH-only system. We find that β-endorphin and ACTH formed an interacting unit, whereas negligible interactions were observed between ACTH molecules in ACTH-only system. Our data suggest that ACTH is not only involved in interaction with β-endorphin but also enhances the stability of mixed oligomers of the entire system

MD simulation of β-end system.

<p>All-atom MD simulation for the duration of 20 ns was performed in explicit solvent using four β-end. A) Snapshots indicating the initial (left) and final (right) states of the β-end system. B) Plot of distances between center of masses (Dij) of peptides within the β-end simulation against time. C) Contact map showing various residues in contact between peptides at t = 20 ns. Significant contacts are observed between amino acid residues in β-end A, β-end B and β-end D. D) A schematic depicting the various h-bonds observed between the peptides during the β-end simulation. The schematic showing C-terminus of β-end D is in contact with N-terminus of β-end A whereas C-terminus of β-end B is in contact with C-terminus β-end A. A salt bridge between E31 of β-end A and K28 of β-end B is represented by red dotted line.</p

Aggregation and colocalization of ACTH and β-end.

<p>A) Fluorescence photomicrographs of the anterior (A–C) and intermediate (D–F) lobes of the pituitary gland showing ACTH (A, D), β-end (B, E), and ACTH-β-end colocalized (C, F) cells. Note that presence of several double-labeled cells (arrows) in anterior as well as intermediate lobes. Scale bar = 50 µm. B) EM images of ACTH and β-end samples incubated for 14 days. The aggregations of the hormones were followed at 37°C at a concentration of 2 mg/ml in the presence of 0.4 mM LMW heparin in 5% D-mannitol (pH 5.5) and PBS under slight agitation. TEM of negative stained samples was performed. Scale bars, 500 nm.</p

MD simulation of ACTH system.

<p>A) Snapshots at the left-hand side indicating initial state (t = 0 ns) and at the right-hand side indicating final state (t = 20 ns) of the simulation. B) Plot of distances between center of masses (Dij) of peptides within the ACTH simulation against time showing large interpeptide distances during simulation. C) Contact map at t = 20 ns snapshot showing residues in contact. Negligible interpeptide contacts are observed suggesting that peptides are not self-assembling <i>in silico</i>.</p