GLUT1 is up-regulated in SIRT6-KO retina.

<p>a) GLUTl immunoreactivity in cross-section of WT and SIRT6-KO mice retina. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL) Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). GLUT1 protein (b) and mRNA levels (c) were determined by Western blot and RT-PCR respectively. β-actin was used as loading control. Data are mean ± SE (n = 6 eyes/group) **p<0.01</p

SIRT6 is active in the mouse retina.

<p>a) H3K56 acetylation is shown by immunofluoescence. b) Representative Western blot showing protein levels of SIRT6 and the acetylation levels of H3K56 and H3K9 in chromatin preparations from WT and KO mice retinas. Total H3 was used for normalization. c) Quantification of the intensity of bands was determined by using the ImageJ and is represented as arbitrary units. Data are mean ± SE (n = 6 eyes/group). **p<0.01, ***p<0.001</p

Retinal functional evaluation.

<p>Representative scotopic (A) and photopic (C) electroretinograms from WT and SIRT6-KO mice at different light intensities (dBs). Plots B and D depict average amplitudes of <i>a</i>-wave and <i>b</i>-wave. Note that the fold decrease of the scotopic <i>a</i>-wave amplitude (8) is greater than the fold decrease of the photopic <i>a</i>-wave amplitude (2,5). Data are mean ± SE (n =  4). **p<0.01, ***p<0.001.</p

Grm6 is down-regulated in SIRT6-KO retinas.

<p>Whole retina mRNA from WT and KO mice was used to profile the expression of several key genes of glutamate receptors involved in the synaptic transmission in an Affymetrix Mouse Gene 2.1 ST DNA microarray. a) Heatmap representing the hierarchical cluster analysis shows the differential expressed mRNAs between WT and SIRT6 KO retinas. The graphic depicts the expression levels of ionotropic AMPA glutamate receptors (Gria1–4), Glutamate receptor, ionotropic kainate (Grik1-2-4-5), Glutamate [NMDA] receptors (Grin1-2a-c) and metabotropic glutamate receptors (Grm1–8). The expression data for the hierarchical clustering image has been row normalized to a range of zero to one with blue representing the row minimum and red representing the row maximum. b) RNA was purified from SIRT6 WT and KO retinas, and Grm6 levels analyzed by RT-PCR. c) immunofluorescence was performed in SIRT6 WT and KO retinas with the indicated antibodies. PKC-alpha was used as a marker for ON bipolar cells. Ganglion Cell Layer (GCL), Inner Plexiform Layer (IPL), Inner nuclear Layer (INL), Outer Plexiform Layer (OPL), Outer Nuclear Layer (ONL), Retinal Pigment Epithelium (RPE). Data are mean ± SE (n = 4) **p<0.01 d) Representative fluorescent images of TUNEL analysis performed in WT and SIRT6 KO retinal sections. Apoptotic nuclei (bright green dots) labeled with fluorescein-dUTP were visualized by fluorescence microscopy. Data are mean ± SE (n  = 3) **p<0.01</p

AMPK-NF-κB Axis in the Photoreceptor Disorder during Retinal Inflammation

<div><p>Recent progress in molecular analysis has revealed the possible involvement of multiple inflammatory signaling pathways in pathogenesis of retinal degeneration. However, how aberrant signaling pathways cause tissue damage and dysfunction is still being elucidated. Here, we focus on 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), originally recognized as a key regulator of energy homeostasis. AMPK is also modulated in response to inflammatory signals, although its functions in inflamed tissue are obscure. We investigated the role of activated AMPK in the retinal neural damage and visual function impairment caused by inflammation. For this purpose, we used a mouse model of lipopolysaccharide-induced inflammation in the retina, and examined the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). During inflammation, activated AMPK in the neural retina was decreased, but AICAR treatment prevented this change. Moreover, the electroretinogram (ERG) a-wave response, representing photoreceptor function, showed visual dysfunction in this model that was prevented by AICAR. Consistently, the model showed shortened photoreceptor outer segments (OSs) with reduced levels of rhodopsin, a visual pigment concentrated in the OSs, in a post-transcriptional manner, and these effects were also prevented by AICAR. In parallel, the level of activated NF-κB increased in the retina during inflammation, and this increase was suppressed by AICAR. Treatment with an NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ) preserved the rhodopsin level during inflammation, suppressing NF-κB. These findings indicated that AMPK activation by AICAR and subsequent NF-κB inhibition had a protective effect on visual function, and that AMPK activation played a neuroprotective role during retinal inflammation.</p></div

Preservation of rhodopsin level by an inhibitor of NF-κB activation, DHMEQ, during inflammation.

<p>(A, B) Immunoblot analysis. The rhodopsin level measured 24 hours after LPS injection was preserved by DHMEQ treatment. (C, D) The shortening of OS length during EIU was avoided by DHMEQ. Relative OS length was measured in the mid-peripheral retina. (E, F) Immunoblot analysis. The level of p-NF-κB p65 was decreased by DHMEQ in the retina 1.5 hours after LPS injection. *P<0.05. **P<0.01. Scale bar, 50 µm. (A, B) All groups, n = 8. (C, D) All groups, n = 5. (E, F) Control, n = 8; EIU with vehicle treatment, n = 8; EIU with AICAR treatment, n = 7. p-NF-κB p65, phosphorylated NF-κB p65.</p

Protective effect of AICAR on the rhodopsin level and OS length during inflammation.

<p>(A, B) Immunoblot analysis. Rhodopsin protein in the retina was decreased during EIU, and this decrease was attenuated by AICAR, 24 hours after LPS injection. (C) Real-time PCR. rhodopsin mRNA was constant 24 hours after LPS injection with or without AICAR treatment. (D) The OS length was shortened during EIU, and this influence was suppressed by AICAR. (E) Relative OS length was measured in the mid-peripheral retina. *P<0.05. **P<0.01. Scale bar, 50 µm. (A–C) Control, n = 4; EIU with vehicle treatment, n = 5; EIU with AICAR treatment, n = 5. (D, E) All groups, n = 6. ONL, outer nuclear layer; IS, inner segment; OS, outer segment.</p

Protective effect of AICAR on visual function during inflammation.

<p>(A–E) ERG data 24 hours after LPS injection. (A) Representative wave responses of scotopic ERG intensity series from an individual mouse. The amplitudes of the a-wave (B) and b-wave (C) were decreased during EIU, but AICAR treatment clearly prevented the decrease. The implicit times of the a-wave (D) and b-wave (E) were prolonged during EIU, but this effect was significantly avoided by AICAR. *P<0.05. **P<0.01. All groups, n = 8.</p

Suppressive effect of AICAR on NF-κB activation during inflammation.

<p>(A, B) Immunoblot analysis. The level of p-NF-κB p65 was increased in the retina 1.5 hours after LPS injection. AICAR significantly blocked the increase of the p-NF-κB p65 level during EIU. *P<0.05. All groups, n = 7. p-NF-κB p65, phosphorylated NF-κB p65.</p