Dynein Light Chain Tctex-Type 1 Modulates Orexin Signaling through Its Interaction with Orexin 1 Receptor

Orexins (OX-A, OX-B) are neuropeptides involved in the regulation of the sleep-wake cycle, feeding and reward, via activation of orexin receptors 1 and 2 (OX1R, OX2R). The loss of orexin peptides or functional OX2R has been shown to cause the sleep disorder, narcolepsy. Since the regulation of orexin receptors remains largely undefined, we searched for novel protein partners of the intracellular tail of orexin receptors. Using a yeast two-hybrid screening strategy in combination with co-immunoprecipitation experiments, we found interactions between OX1R and the dynein light chains Tctex-type 1 and 3 (Dynlt1, Dynlt3). These interactions were mapped to the C-terminal region of the dynein light chains and to specific residues within the last 10 amino acids of OX1R. Hence, we hypothesized that dynein light chains could regulate orexin signaling. In HEK293 cells expressing OX1R, stimulation with OX-A produced a less sustained extracellular signal-regulated kinases 1/2 (ERK1/2) activation when Dynlt1 was co-expressed, while it was prolonged under reduced Dynlt1 expression. The amount of OX1R located at the plasma membrane as well as the kinetics and extent of OX-A-induced internalization of OX1R (disappearance from membrane) were not altered by Dynlt1. However, Dynlt1 reduced the localization of OX1R in early endosomes following initial internalization. Taken together, these data suggest that Dynlt1 modulates orexin signaling by regulating OX1R, namely its intracellular localization following ligand-induced internalization

Lipid droplets under stressful conditions

Lipid droplets (LDs) are phylogenetically conserved and ubiquitous organelles with many cellular functions. In the last two decades, our understanding of LD biology and of their roles in physiological processes has increased dramatically. In addition, increasing evidence suggests that LDs are highly involved in inflammatory processes, and in metabolic disorders such as type 2 diabetes mellitus (T2DM). Despite such advancement, many aspects of LD biology and of their roles in health and disease remain unknown.The core of LDs is highly enriched with neutral lipids and these can be mobilized to provide metabolic energy. The phospholipid monolayer surrounding the LD core is associated with a wide variety of proteins, including structural and signaling proteins, as well as metabolic enzymes. While LDs may be induced by physiological stimuli such as dietary fatty acids, they can also be formed under stressful conditions, in the absence of such fatty acids. However, exactly how cellular stress leads to LD accumulation remains unclear. Our main objective is to understand the regulation of LD formation under stressful conditions, specifically oxidative stress, inflammation, and metabolic stress. We first investigated LDs in cells exposed to environmental stressors, namely cytotoxic metallic nanoparticles and reactive oxygen species. LD formation and expression of perilipin-2, a key structural LD protein, were highly increased in rodent cells exposed to these stress agents. Interestingly, supplementation with antioxidant N-acetyl cysteine or pharmacological inhibition of p38 mitogen activated protein kinase (MAPK) reduced stress-induced LD accumulation, suggesting that oxidative stress and p38 MAPK activation play a role in the induction of LD formation. Inflammatory leukocytes and macrophages contain a large number of LDs. While this phenomenon has been widely investigated in peripheral immune cells, its explanation remains elusive in immune cells of the central nervous system. We therefore investigated LD dynamics and regulation in microglia, the resident immune cells in the brain. We found that stimulation of microglia with toll-like receptor 4 (TLR4) agonist, lipopolysaccharides (LPS), increased LD formation and perilipin-2 expression in an Akt and p38 MAPK-dependent manner. Interestingly, LPS-induced LDs extensively colocalized with cytosolic phospholipase A2-α (cPLA2-α), a key enzyme involved in the synthesis of eicosanoids, which are inflammatory lipid mediators. Collectively, these findings imply that LD formation may contribute to increased eicosanoid synthesis in activated microglia and could be microglial biomarkers of inflammation in the central nervous system. To gain a better insight into the role of LDs in human pathology, we sought to examine alterations in LD metabolism in pancreatic tissue obtained from T2DM and obese individuals. Immunohistochemical studies revealed increased islet and extra-islet perilipin-2 expression in tissues from lean or obese T2DM donors, but not in non-T2DM obese donors, suggesting that the diabetic status, but not the obesity status, is a requirement for increasing perilipin-2 expression and LD formation. Gene expression analysis by RT-qPCR confirmed the increase in perilipin-2 expression and revealed significant alterations in several genes related to islet function, metabolism and antioxidant defense. These alterations seem to be consistently associated with obesity and T2DM and imply an adaptive and compensatory response to insulin resistance and metabolic stress. In summary, our studies show that LDs are an integral part of the adaptive cellular response to oxidative, inflammatory and metabolic stress. Perhaps, the most important challenge in LD research in the upcoming decade will be to determine how the subcellular lipid and protein composition of this organelle affects its function in different cells.Les gouttelettes lipidiques (GL) sont des organites phylogénétiquement conservées et impliquées dans plusieurs fonctions cellulaires. Durant les deux dernières décennies, notre compréhension des rôles biologiques et physiologiques des GL a augmenté de manière draconienne. Plusieurs observations suggèrent fortement que les GL jouent un rôle important dans l'inflammation, ainsi que dans les désordres métaboliques tels que le diabète de type 2 (DT2). Malgré cette avancée, plusieurs aspects de la biologie des GL et de leurs rôles dans des maladies demeurent méconnus.Le centre des GL est riche en lipides neutres qui peuvent se mobiliser et servir comme source d'énergie. La couche phospholipidique entourant le centre de la GL est associée à plusieurs protéines et enzymes métaboliques. Bien que les GL puissent être induites par des acides gras, elles peuvent aussi l'être dans des conditions de stress. Par contre, les mécanismes de l'accumulation de GL par des conditions de stress ne sont pas encore bien compris. Notre objectif principal est de comprendre la régulation de la formation de GL par le stress oxydatif, l'inflammation et le stress métabolique. Premièrement, nous avons investigué les GL dans des cellules exposées à des stresseurs tels que des nanocrystaux métalliques et des dérivés réactifs d'oxygène. La formation de GL et l'expression de perilipin-2, qui est une protéine structurelle des GL, ont tous deux augmenté dans les cellules stressées. De plus, une supplémentation en antioxydant (n-acétylcystéine) ou un traitement avec un inhibiteur de p38 MAPK a réduit l'accumulation de GL causée par le stress. Ces observations suggèrent que le stress oxydatif et p38 MAPK jouent un rôle dans l'accumulation de GL dans des cellules stressées. Il est bien connu que les leucocytes et macrophages qui sont engagés dans l'inflammation contiennent une grande quantité de GL. Même si ce phénomène a bien été exploré dans les cellules immunitaires périphériques, il reste inexploré dans le système nerveux central (SNC). Ce faisant, nous avons investigué la dynamique et la régulation des GL dans les microglies, les cellules résidentes immunitaires dans le cerveau. Nous avons trouvé que dans les microglies stimulées avec les lipopolysaccharides (LPS), les GL et l'expression de perilipin-2 ont augmenté d'une manière dépendante de l'activation de l'Akt et p38 MAPK. Dans ces cellules activées, la phospholipase cytosolique A2-α (PLC A2-α), une enzyme fonctionnant dans la synthèse d'éicosanoides, des médiateurs lipidiques inflammatoires, colocalisait avec les GL. Ensemble, ces résultats indiquent que la formation de GL pourrait contribuer à la synthèse d'éicosanoides dans les microglies activées et servir de biomarqueurs d'inflammation dans le SNC.Pour mieux comprendre le rôle des GL dans la pathologie humaine, nous les avons examinées dans des tissues pancréatiques provenant de patients obèses ou diabétiques T2. Nos études immunohistochimiques ont révélé une augmentation de perilipin-2 dans les îlots de Langerhans chez les patients diabétiques obèses ou maigres, mais pas dans ceux de patients non-diabétiques. Ceci suggère que le DT2, mais non l'obésité, est requis pour une augmentation de perilipin-2 dans le pancréas. L'analyse d'expression de gènes par RT-PCR a confirmé l'augmentation de perilipin-2 observé antérieurement dans les îlots et a également révélé des altérations dans des gènes reliés aux fonctions des îlots, au métabolisme, et aux défenses anti-oxydantes. Ces changements, qui sont souvent associés à l'obésité et au DT2, constituent un mécanisme d'adaptation à la résistance à l'insuline et au stress métabolique.Pour résumer, nos études démontrent que l'accumulation de GL fait partie intégrante de l'adaptation des cellules au stress. Durant la prochaine décennie, le plus grand obstacle dans la recherche sur les GL sera de déterminer comment la composition lipidique ou protéinique de ces organites affecte leurs fonctions biologiques

Levels of OX1R in resting conditions at the plasma membrane in presence or absence of Dynlt1.

<p>Control refers to a different transfection for each experiment. Empty pCS2, 10 nM or 20 nM negative control siRNA were used respectively as control for the transfection of pCS2-Dynlt1, <i>Dynlt1</i> siRNA or a mix of <i>Dynlt1</i> and <i>Dynlt3</i> siRNAs.</p

Dynlt1 alters the intracellular localization of OX1R following ligand-induced internalization.

<p>(<b>A</b>) HEK293 cells were transfected with pEGFP-N1-OX1R and stimulated with OX-A for the indicated times. IB analysis of whole cell lysates shows that ERK1/2 phosphorylation can be achieved after stimulation of this OX1R-GFP fusion protein with OX-A. (<b>B</b>) HEK293 cells were transfected with pCS2-Myc-Dynlt1 and pEGFP-N1-OX1R or corresponding empty vector. Whole-cell lysates were subjected to IP with an anti-GFP antibody to immunoprecipitate OX1R-GFP and anti-Myc antibody to reveal Myc-Dynlt1. Equal transfection of Myc-Dynlt1 was verified by IB with anti-Myc antibody on cell extracts (input). (<b>C, D</b>) HEK293 cells stably expressing OX1R-GFP were transfected with control plasmid (<b>C</b>) or pCS2-FLAG-Dynlt1 (<b>D</b>), stimulated with OX-A for 5–30 min or left un-stimulated (0 min) and analyzed by fluorescent microscopy. Cells were labeled with an antibody specific for a marker of early endosomes, EEA1. Maximum co-localization of green pixels with red pixels occurs after 15 min OX-A in control conditions. However, when over-expressing Dynlt1, co-localization is rather similar across OX-A stimulation times. Left panels: green GFP fluorescence; OX1R, middle panels: EEA1 labeling, red ALEXA 568 secondary antibody; early endosomes, and right panels: the merged images. (<b>E</b>) Magnification of merged fields from panels C and D (white squares) after 15 min OX-A without or with Dynlt1 over-expression. White arrows indicate OX1R-GFP signal (green) not co-localized with endosome marker (red). (<b>F</b>) Manders' co-localization coefficient for green channel (OX1R-GFP) was generated on images that were co-localized using Openlab software. Manders' co-localization coefficient (OX1R): co-localized green and red pixels divided by total number of green pixels over threshold. Results represent the mean ± SEM of triplicates from one experiment, where at least 5 fields with 1–3 cells in each were analyzed for each time point. This experiment was done twice with similar results (three times for the 0 and 15 min time points). The co-localization of OX1R-GFP with the EEA1 marker for endosomes, after 15 min OX-A, is decreased upon Dynlt1 over-expression. *: p<0.05 when compared to control transfection (without Dynlt1) after 15 min OX-A.</p

Interaction of OX1R and Dynlt1 in mammalian cells.

<p>HEK293 cells were transfected with expression vectors for Myc-Dynlt1 and V5-OX1R (0.5 or 2.0 µg DNA, full-length receptor) or corresponding empty vector. Whole-cell lysates were subjected to IP using an anti-V5 antibody and protein G-sepharose beads, followed by IB with anti-V5 or anti-Myc antibodies against V5-OX1R and Myc-Dynlt1, respectively. Equal transfection of Myc-Dynlt1 was verified by IB with anti-Myc antibody on cell extracts (input). The experiment was repeated 5 times, with similar results.</p

Modulation of OX1R-mediated signaling by Dynlt1.

<p>(<b>A</b>) HEK293 cells were transfected with a V5-OX1R or V5-OX1R Δ364–416 expression vector and stimulated with 500 nM OX-A for the indicated times. Protein extracts were analyzed by SDS-PAGE and Western blotting with anti-phospho-ERK1/2 and anti-ERK1/2 antibodies. ERK1/2 are quickly phosphorylated after OX-A stimulation and OX1R CTD is essential to this response. V5-OX1R Δ364–416, V5-OX1R lacking its CTD. (<b>B–D</b>) Transfected HEK293 cells were stimulated with 100 nM OX-A for the indicated times and processed as in (A). (<b>B</b>) Co-expression of Dynlt1 leads to a less sustained ERK1/2 activation in response to OX-A. Blots are from a representative experiment. The graph shows a combination of 4 independent experiments. ***: p<0.001 vs data without Dynlt1 transfected (ANOVA followed by post-hoc analysis at the different times). (<b>C</b>) Down-regulation of Dynlt1 by a siRNA (10 nM, expression reduced by 88%) leads to a sustained activation of the ERK1/2 pathway in response to OX-A. This experiment was repeated twice, each in duplicates, with similar results. The blots and the graph present the results from one of these experiments. (<b>D</b>) Dynlt1 does not lead to a less sustained ERK1/2 activation in response to OX-A when co-expressed with OX1R mutant T409A, T412A. This experiment was repeated twice, each in triplicates, with similar results. The blots and the graph are from one of these experiments.</p

Regions of orexin receptors involved in their interaction with Dynlt1 and Dynlt3.

<p>(<b>A</b>) Identification of a putative bipartite Dynlt1-binding motif in orexin receptors. The proximal portion of the structure shown here, located in the third intracellular loop of orexin receptors, is not included in the soluble orexin receptors CTD containing the distal part and used for yeast two-hybrid assays (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone-0026430-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone-0026430-g003" target="_blank">3</a>). Amino acid numbering refers to mouse sequences. Amino acids of the consensus delineated from other Dynlt1-binding proteins are shown in bold, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone.0026430-Mok1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026430#pone.0026430-Sugai1" target="_blank">[31]</a>. In the OX1R CTD mutant, two conserved Thr were mutated to Ala (409 and 412, numbering from full-length OX1R). (<b>B, C, D</b>) β-galactosidase (top panels) and <i>HIS3</i> (bottom panels) assays were performed on yeast transformed with plasmids expressing different combinations of orexin receptor and Dynlt1/Dynlt3. Interactions of OX1R CTD with Dynlt1 and Dynlt3 are reduced when Thr 409 and 412 of OX1R CTD are mutated into Ala. Deleting the extra amino acids of OX2R CTD favors its interaction with Dynlt1, while deleting the next 10 amino acids (comprising the distal part of the Dynlt1-binding motif) abolishes this effect. (<b>E</b>) Summary of OX1R CTD and OX2R CTD constructs tested and their relative interaction strength with Dynlt1 and Dynlt3. OX1R CTD T409, 412A, OX1R CTD with T409 and T412 mutated into alanine residues; OX1R CTD Δ407–416, OX1R CTD lacking the last 10 a.a.; OX1R CTD Δ397–416, OX1R CTD lacking the last 20 a.a.; OX1R CTD Δ387–416, OX1R CTD lacking the last 30 a.a.; OX2R CTD Δ433–460, OX2R CTD lacking the extra 28 a.a. compared to OX1R CTD; OX2R CTD Δ423–460, OX2R CTD lacking the last 38 a.a.; OX2R CTD Δ413–460, OX2R CTD lacking the last 48 a.a.; OX2R CTD Δ403–460, OX2R CTD lacking the last 58 a.a. ND, not determined. Experiments were performed 3 times and the average is presented. **: p<0.01 vs transformation with wild-type OX1R CTD or OX2R CTD.</p

Agonist-induced internalization of OX1R in presence or down-regulation of Dynlt1/Dynlt3.

<p>Cell surface detection of OX1R was measured by ELISA. HEK293 cells were transfected with either 0.1 (A) or 0.25 µg (B, C, D) control pSG5-V5-His or pSG5-V5-His-OX1R, in combination with: (<b>A</b>) pCS2-Myc or pCS2-Myc-Dynlt1, (<b>B</b>) 10 nM of either control siRNA or <i>Dynlt1</i> siRNA (80% down-regulation), or (<b>C, D</b>) either 20 nM control siRNA or a combination of <i>Dynlt1</i> and <i>Dynlt3</i> siRNAs at 10 nM each (75% and 80% down-regulation, respectively). Cells were then treated with OX-A for up to 30 min. Normalized OD values refer to OD values that have been corrected (background-subtracted) and then expressed relative to basal conditions (% of value before stimulation for each condition). For all panels, there is maximum OX1R at the membrane at basal conditions (0 min; without OX-A) and the receptor is internalized (represented by loss at the membrane) after OX-A treatment. Results represent the mean ± SEM of 3 experiments, each performed in triplicate (except for panel D; results represent the mean ± SEM of 2 experiments). There is no statistical significance for the effect of over-expressing or down-regulating Dynlt1 and/or Dynlt3 as assessed by two-way ANOVA.</p