NLRP3 Inflammasome Priming in the Retina of Diabetic Mice Requires REDD1-Dependent Activation of GSK3ß.

Purpose: Inflammasome activation has been implicated in the development of retinal complications caused by diabetes. This study was designed to identify signaling events that promote retinal NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation in response to diabetes. Methods: Diabetes was induced in mice by streptozotocin administration. Retinas were examined after 16 weeks of diabetes. Human MIO-M1 Müller cells were exposed to hyperglycemic culture conditions. Genetic and pharmacological interventions were used to interrogate signaling pathways. Visual function was assessed in mice using a virtual optomotor system. Results: In the retina of diabetic mice and in Müller cell cultures, NLRP3 and interleukin-1ß (IL-1ß) were increased in response to hyperglycemic conditions and the stress response protein Regulated in Development and DNA damage 1 (REDD1) was required for the effect. REDD1 deletion prevented caspase-1 activation in Müller cells exposed to hyperglycemic conditions and reduced IL-1ß release. REDD1 promoted nuclear factor κB signaling in cells exposed to hyperglycemic conditions, which was necessary for an increase in NLRP3. Expression of a constitutively active GSK3ß variant restored NLRP3 expression in REDD1-deficient cells exposed to hyperglycemic conditions. GSK3 activity was necessary for increased NLRP3 expression in the retina of diabetic mice and in cells exposed to hyperglycemic conditions. Müller glia-specific REDD1 deletion prevented increased retinal NLRP3 levels and deficits in contrast sensitivity in diabetic mice. Conclusions: The data support a role for REDD1-dependent activation of GSK3ß in NLRP3 inflammasome transcriptional priming and in the production of IL-1ß by Müller glia in response to diabetes.

PERK/ATF4-dependent expression of the stress response protein REDD1 promotes proinflammatory cytokine expression in the heart of obese mice.

Endoplasmic reticulum (ER) stress and inflammation are hallmarks of myocardial impairment. Here, we investigated the role of the stress response protein regulated in development and DNA damage 1 (REDD1) as a molecular link between ER stress and inflammation in cardiomyocytes. In mice fed a high-fat high-sucrose (HFHS, 42% kcal fat, 34% sucrose by weight) diet for 12 wk, REDD1 expression in the heart was increased in coordination with markers of ER stress and inflammation. In human AC16 cardiomyocytes exposed to either hyperglycemic conditions or the saturated fatty acid palmitate, REDD1 expression was increased coincident with ER stress and upregulated expression of the proinflammatory cytokines IL-1ß, IL-6, and TNFα. In cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions, pharmacological inhibition of the ER kinase protein kinase RNA-like endoplasmic reticulum kinase (PERK) or knockdown of the transcription factor ATF4 prevented the increase in REDD1 expression. REDD1 deletion reduced proinflammatory cytokine expression in both cardiomyocytes exposed to hyperglycemic/hyperlipidemic conditions and in the hearts of obese mice. Overall, the findings support a model wherein HFHS diet contributes to the development of inflammation in cardiomyocytes by promoting REDD1 expression via activation of a PERK/ATF4 signaling axis.NEW & NOTEWORTHY Interplay between endoplasmic reticulum stress and inflammation contributes to cardiovascular disease progression. The studies here identify the stress response protein known as REDD1 as a missing molecular link that connects the development of endoplasmic reticulum stress with increased production of proinflammatory cytokines in the hearts of obese mice.

Spleen Tyrosine Kinase Contributes to Müller Glial Expression of Proangiogenic Cytokines in Diabetes.

Purpose: Neuroglial dysfunction occurs early in the progression of diabetic retinopathy. In response to diabetes or hypoxia, Müller glia secrete cytokines and growth factors that contribute to disease progression. This study was designed to examine common signaling pathways activated in Müller glia by both type 1 and pre-/type 2 diabetes. Methods: RiboTag (Pdgfra-cre;HA-Rpl22) mice were used to compare the impact of streptozotocin (STZ) and a high-fat, high-sucrose (HFHS) diet on ribosome association of mRNAs in Müller glia by RNA sequencing analysis. Human MIO-M1 Müller cells were exposed to either hyperglycemic or hypoxic culture conditions. Genetic manipulation and pharmacologic inhibition were used to interrogate signaling pathways. Results: Association of mRNAs encoding triggering receptor expressed on myeloid cells 2 (TREM2), DNAX-activating protein 12 kDa (DAP12), and colony stimulating factor 1 receptor (CSF1R) with ribosomes isolated from Müller glia was upregulated in both STZ diabetic mice and mice fed an HFHS diet. The TREM2/DAP12 receptor-adaptor complex signals in coordination with CSF1R to activate spleen tyrosine kinase (SYK). SYK activation was enhanced in the retina of diabetic mice and in human MIO-M1 Müller cell cultures exposed to hyperglycemic or hypoxic culture conditions. DAP12 knockdown reduced SYK autophosphorylation in Müller cells exposed to hyperglycemic or hypoxic conditions. SYK inhibition or DAP12 knockdown suppressed hypoxia-induced expression of the transcription factor hypoxia-inducible factor 1⍺ (HIF1⍺), as well as expression of vascular endothelial growth factor and angiopoietin-like 4. Conclusions: The findings support TREM2/DAP12 receptor-adaptor complex signaling via SYK to promote HIF1α stabilization and increased angiogenic cytokine production by Müller glia.

REDD1 Ablation Attenuates the Development of Renal Complications in Diabetic Mice.

Chronic hyperglycemia contributes to development of diabetic kidney disease by promoting glomerular injury. In this study, we evaluated the hypothesis that hyperglycemic conditions promote expression of the stress response protein regulated in development and DNA damage response 1 (REDD1) in the kidney in a manner that contributes to the development of oxidative stress and renal injury. After 16 weeks of streptozotocin-induced diabetes, albuminuria and renal hypertrophy were observed in wild-type (WT) mice coincident with increased renal REDD1 expression. In contrast, diabetic REDD1 knockout (KO) mice did not exhibit impaired renal physiology. Histopathologic examination revealed that glomerular damage including mesangial expansion, matrix deposition, and podocytopenia in the kidneys of diabetic WT mice was reduced or absent in diabetic REDD1 KO mice. In cultured human podocytes, exposure to hyperglycemic conditions enhanced REDD1 expression, increased reactive oxygen species (ROS) levels, and promoted cell death. In both the kidney of diabetic mice and in podocyte cultures exposed to hyperglycemic conditions, REDD1 deletion reduced ROS and prevented podocyte loss. Benefits of REDD1 deletion were recapitulated by pharmacological GSK3ß suppression, supporting a role for REDD1-dependent GSK3ß activation in diabetes-induced oxidative stress and renal defects. The results support a role for REDD1 in diabetes-induced renal complications.