Deletion of miR-150 Exacerbates Retinal Vascular Overgrowth in High-Fat-Diet Induced Diabetic Mice

Diabetic retinopathy (DR) is the leading cause of blindness among American adults above 40 years old. The vascular complication in DR is a major cause of visual impairment, making finding therapeutic targets to block pathological angiogenesis a primary goal for developing DR treatments. MicroRNAs (miRs) have been proposed as diagnostic biomarkers and potential therapeutic targets for various ocular diseases including DR. In diabetic animals, the expression levels of several miRs, including miR-150, are altered. The expression of miR-150 is significantly suppressed in pathological neovascularization in mice with hyperoxia-induced retinopathy. The purpose of this study was to investigate the functional role of miR-150 in the development of retinal microvasculature complications in high-fat-diet (HFD) induced type 2 diabetic mice. Wild type (WT) and miR-150 null mutant (miR-150-/-) male mice were given a HFD (59% fat calories) or normal chow diet. Chronic HFD caused a decrease of serum miR-150 in WT mice. Mice on HFD for 7 months (both WT and miR-150-/-) had significant decreases in retinal light responses measured by electroretinograms (ERGs). The retinal neovascularization in miR-150-/--HFD mice was significantly higher compared to their age matched WT-HFD mice, which indicates that miR-150 null mutation exacerbates chronic HFD-induced neovascularization in the retina. Overexpression of miR-150 in cultured endothelial cells caused a significant reduction of vascular endothelial growth factor receptor 2 (VEGFR2) protein levels. Hence, deletion of miR-150 significantly increased the retinal pathological angiogenesis in HFD induced type 2 diabetic mice, which was in part through VEGFR2

A new functional role for mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in the circadian regulation of L-type voltage-gated calcium channels in avian cone photoreceptors.

In the retina, the L-type voltage-gated calcium channels (L-VGCCs) are responsible for neurotransmitter release from photoreceptors and are under circadian regulation. Both the current densities and protein expression of L-VGCCs are significantly higher at night than during the day. However, the underlying mechanisms of circadian regulation of L-VGCCs in the retina are not completely understood. In this study, we demonstrated that the mechanistic/mammalian target of rapamycin complex (mTORC) signaling pathway participated in the circadian phase-dependent modulation of L-VGCCs. The activities of the mTOR cascade, from mTORC1 to its downstream targets, displayed circadian oscillations throughout the course of a day. Disruption of mTORC1 signaling dampened the L-VGCC current densities, as well as the protein expression of L-VGCCs at night. The decrease of L-VGCCs at night by mTORC1 inhibition was in part due to a reduction of L-VGCCα1 subunit translocation from the cytosol to the plasma membrane. Finally, we showed that mTORC1 was downstream of the phosphatidylionositol 3 kinase-protein kinase B (PI3K-AKT) signaling pathway. Taken together, mTORC1 signaling played a role in the circadian regulation of L-VGCCs, in part through regulation of ion channel trafficking and translocation, which brings to light a new functional role for mTORC1: the modulation of ion channel activities

The mTORC1 pathway is downstream of PI3K-AKT.

<p>Chick embryos (E11) were entrained in LD cycles for 7 days <i>in ovo</i>, and retinas were dissected, cultured, and kept in DD. On the second day of DD, cultured retinas were treated with 0.1% DMSO (control) or various inhibitors for 2 hr prior to harvest for immunoblotting at CT 4 and CT 16. (A) The phosphorylation of AKT at thr308 (pAKT_thr308) was significantly higher in the control and rapamycin treated cells at CT 16 compared to other groups. “n.s.” indicates that there is no statical difference between the two CT 16 groups. * indicates statistical differences. (B) The phosphorylation of S6 (pS6), a downstream target of mTORC1, is significantly higher in the control at CT 16 (*) compared to all other groups. The level of pS6 in the control at CT 4 (#) is significantly higher than groups treated with rapamycin or PP242. (C) The levels of pAKT_thr308 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. The levels of pAKT_thr308 are significantly lower in both groups (CT 4 and CT 16) treated with LY294002 (#). (D) The levels of pS6 are significantly higher in the control at CT 16 (*) and PD98059 treated group at CT 16 (*) compared to all other groups. (E) The level of di-phosphorylated ERK (pERK) is significantly higher in the control at CT 16 (*), and there is no statistical difference between the control at CT 16 and LY294002 treated group at CT 16. The pERK levels in both PD98059 treated groups (CT 4 and CT 16) are significantly lower (#) compared to all other groups. (F) Treatment with rapamycin or PP242 does not affect the circadian rhythm of pERK, in which the pERK level is significantly higher at night (CT 16, *) compared to the day time level (CT 4). *, ^#<i>p</i><0.05.</p

mTORC1 inhibition dampens the circadian rhythm of L-VGCCα1D.

<p>Retinal cells were cultured on glass coverslips and entrained to 12 hr LD cycles for four days in vitro and kept in DD. On the second day of DD, cells were treated with rapamycin at CT 2 and CT 14 for 2 hr followed by fixation at CT 4 and CT 16. After washing and blocking, cells were processed for L-VGCCα1D immunofluorescent staining. (A) Epifluorescent photos from the control (top panel) and rapamycin treated (lower panel) groups. The photographs shown here were slightly over-exposed to provide clearer images but were not used for statistical analysis in (B). The arrowheads indicate the cone photoreceptors. (B) The fluorescence intensity of L-VGCCα1D was significantly higher at CT 16 (control) compared to all other groups. Each group has at least 15 cells from 4 different trials. *<i>p</i><0.05.</p

mTORC1 inhibitor dampens the circadian rhythm of L-VGCC currents.

<p>L-VGCCs were recorded from cultured chick cone photoreceptors on the sixth day of LD entrainment during the day (ZT 4-7) or at night (ZT 16-19). (A) Representative day (ZT 4-7) and night (ZT 16-19) control L-VGCC current traces from cells treated with 0.1% DMSO; (A) ramp command, (B) step commands. (C) Two representative traces from cells that were treated with rapamycin (1 µM) for 2 hr prior to recordings. (D) and (E) The average current-voltage (I–V) relationships are shown in current density (pA/pF) and step-voltage (mV). * indicates that the control group at ZT 16-19 is significantly different from the other groups. (F) The maximal current densities were elicited at 0 mV. * indicates that the L-VGCC current density recorded at night (control, ZT 16-19; n=23) is significantly higher than those recorded during the day (control, ZT 4-7; n=21), rapamycin treated cells recorded during the day (ZT 4-7; 1 µM n=16; 10 µM n=13) and night (ZT 16-19; 1 µM n=18; 10 µM n=13). * indicates that the L-VGCC current density recorded from the control cells at night (control, ZT16-19) is significantly higher than all other groups. *<i>p</i><0.05.</p

mTOR signaling regulates the circadian phase-dependent modulation of L-VGCCs.

<p>(A) Representative L-VGCC current traces from cells treated with 0.1% DMSO (control) or PP242 (100 nM or 400 nM) are shown. (B) The average current-voltage (I–V) relationships from cells treated with 0.1% DMSO (control) or PP242 (100 nM) are shown in current density (pA/pF) and step-voltage (mV). (C) The maximal current densities were elicited at 0 mV of the step command. * indicates that the L-VGCC current density of the controls recorded at night (ZT 16-19) is significantly higher than control and PP242 (100 nM) treated cells recorded during the day (ZT 4-7). PP242-treated cells that were recorded at night (ZT 16-19) have no statistical difference (n.s.) in L-VGCC current densities when compared to PP242-treated cells recorded during the day (ZT 4-7) or the controls recorded at night (ZT 16-19). Each group had at least 15 cells. (D) The average current-voltage (I–V) relationships from cells treated with 0.1% DMSO (control) or PP242 (400 nM) are shown in current density (pA/pF) and step-voltage (mV). * indicates that the L-VGCC current density of the controls recorded at night (ZT 16-19) is significantly higher than the other three groups. (E) Treatment with PP242 at 400 nM significantly dampened the circadian rhythm of maximal L-VGCC current densities. Each group had at least 15 cells. * indicates that the L-VGCC current density of the control group at night (ZT 16-19) is significantly higher than the other three groups. (F) Treatment with either rapamycin (10µM, 1µM) or PP242 (400nM) does not inhibit calcineurin activity compared to the control (0.1% DMSO) cultures. n=6 for each group. *<i>p</i><0.05.</p

A schematic model summarizes the complex cell signaling in the circadian regulation of L-VGCCs.

<p>The circadian clock in the photoreceptor regulates the mRNA levels and protein expression of the channel forming L-VGCCα1 subunits. The circadian clock also regulates the activities / phosphorylation states of cell signaling molecules. In part, the complex cell signaling involves the trafficking, translocation, plasma membrane insertion, and/or membrane retention of L-VGCCα1. This model is based on our previous observations [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073315#B37" target="_blank">37</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073315#B38" target="_blank">38</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073315#B40" target="_blank">40</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073315#B45" target="_blank">45</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073315#B66" target="_blank">66</a>], as well as the current results.</p

Inhibition of mTORC1 causes a circadian phase-dependent decrease in the protein expression and plasma membrane distribution of L-VGCCα1D.

<p>Chick embryos (E11) were entrained in LD cycles for 7 days <i>in ovo</i>, and retinas were dissected, cultured, and kept in DD. On the second day of DD, cultured retinal cells were treated with 0.1% DMSO (control), rapamycin, or PP242 for 2 hr prior to harvest at CT 4 and CT 16. (A) In the whole cell lysate, the total protein expression of L-VGCCα1D was significantly higher at night (CT 16) of the control compared to all other groups. Treatment with either rapamycin or PP242 dampened the circadian rhythm of L-VGCCα1D protein levels (B1-B3). After treatment with DMSO (control) or rapamycin for 2 hr, the distribution of L-VGCCα1D on the plasma membrane or cytosol was analyzed using biotinylation assays (B1). Representative blots of L-VGCCα1D from the cytosolic compartment (cytosol) and membrane-bound fraction (membrane). Total ERK served as loading control (B2). In the membrane-bound fraction, the L-VGCCα1D was significantly higher in control cells harvested at CT 16 compared to other groups (B3). In the cytosolic compartment (cytosol), there was no difference in the quantity of L-VGCCα1D among all groups. *<i>p</i><0.05.</p

mTORC1 signaling is under circadian control.

<p>On the second day of DD after circadian entrainment to 12: 12 hr LD cycles for 7 days <i>in ovo</i>, intact retinas were collected at 6 different circadian time points (CT 0, 4, 8, 12, 16, and 20) for immunoblotting analysis. (A) Phosphorylation of mTORC1 at ser2448 (pTORC1_ser2448) showed a circadian rhythm with its peak at CT 12. (B) The total amount of mTORC1 (total TORC1) and mTORC1 phosphorylation at ser2481 (pTORC1_ser2481) did not display circadian rhythmicity. (C) Phosphorylation of the downstream target of mTORC1, p70-S6 kinase 1(pp70S6K), displayed a circadian rhythm with a peak at CT 12 (upper panel), while total p70S6K (lower panel) remained constant. (D) Phosphorylation of S6 ribosomal protein (pS6), the downstream target of p70S6 kinase, exhibited a circadian rhythm with a peak at CT 12 (upper panel), with total S6 protein (lower panel) constant throughout the day. * indicates a statistical significance at CT 12 compared to CT 0, CT4, or CT8. *<i>p</i><0.05.</p