Chronic neuroinflammation is a common feature of the ageing brain and some neurodegenerative disorders. However, the molecular and cellular mechanisms underlying the regulation of innate immunity in the central nervous system remain elusive. Here we show that the astrocytic dopamine D2 receptor (DRD2) modulates innate immunity through αB-crystallin (CRYAB), which is known to suppress neuroinflammation. We demonstrate that knockout mice lacking Drd2 showed remarkable inflammatory response in multiple central nervous system regions and increased the vulnerability of nigral dopaminergic neurons to neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. Astrocytes null for Drd2 became hyper-responsive to immune stimuli with a marked reduction in the level of CRYAB. Preferential ablation of Drd2 in astrocytes robustly activated astrocytes in the substantia nigra. Gain- or loss-of-function studies showed that CRYAB is critical for DRD2-mediated modulation of innate immune response in astrocytes. Furthermore, treatment of wild-type mice with the selective DRD2 agonist quinpirole increased resistance of the nigral dopaminergic neurons to MPTP through partial suppression of inflammation. Our study indicates that astrocytic DRD2 activation normally suppresses neuroinflammation in the central nervous system through a CRYAB-dependent mechanism, and provides a new strategy for targeting the astrocyte-mediated innate immune response in the central nervous system during ageing and disease.

MicroRNAs (miRNAs) modulate complex physiological and pathological processes by repressing expression of multiple components of cellular regulatory networks. Here we demonstrate that miRNAs encoded by the miR-23∼27∼24 gene clusters are enriched in endothelial cells and highly vascularized tissues. Inhibition of miR-23 and miR-27 function by locked nucleic acid-modified anti-miRNAs represses angiogenesis in vitro and postnatal retinal vascular development in vivo. Moreover, miR-23 and miR-27 are required for pathological angiogenesis in a laser-induced choroidal neovascularization mouse model. MiR-23 and miR-27 enhance angiogenesis by promoting angiogenic signaling through targeting Sprouty2 and Sema6A proteins, which exert antiangiogenic activity. Manipulating miR-23/ 27 levels may have important therapeutic implications in neovascular age-related macular degeneration and other vascular disorders.blindness | MAP kinase signaling | semaphorins | Akt | proangiogenic T he growth of blood vessels through angiogenesis is a delicately controlled process that involves endothelial cell (EC) activation, proliferation, migration, and maturation (1). Physiological angiogenesis is required for normal vascular development as well as vascular homeostasis during adulthood. Pathological angiogenesis, commonly induced by tissue ischemia or inflammation, underlies numerous vascular disorders, such as age-related macular degeneration (AMD), a leading cause of blindness in the elderly (2). Choroidal neovascularization (CNV), which involves abnormal growth of blood vessels in the back of the eye, is a hallmark of neovascular AMD (3). Although the pathogenic mechanisms underlying AMD are still largely unknown, vascular endothelial growth factor (VEGF) has been shown to play a causal role in the development of CNV (4). Anti-VEGF agents have demonstrated efficacy in treating CNV in neovascular AMD (5, 6) but have limited efficacy and potential side effects (7,8).Recent studies have revealed important roles for microRNAs (miRNAs) in cardiovascular diseases and other disorders (9). miRNAs are small noncoding RNAs that negatively regulate gene expression by inducing mRNA degradation or inhibiting translation through binding to the 3′ untranslated region (3′UTR) of target mRNAs (10). Often, miRNAs modulate broad collections of mRNAs encoding multiple components of complex biological pathways. Several miRNAs have been implicated in angiogenesis (11,12). A group of miRNAs has also been shown to be substantially decreased in a laser-induced CNV model (13).The miR-23∼27∼24 clusters are highly expressed in ECs (14-17). Two miR-23∼27∼24 clusters exist in the vertebrate genome: an intergenic miR-23a∼27a∼24-2 cluster and an intronic miR23b∼27b∼24-1 cluster. Members of these clusters are involved in cell cycle control, proliferation, and differentiation of various cell types (18). Here, we show that inhibition of miR-23/27 impairs angiogenesis in vitro and postnatal retinal vascular development in vivo. Moreover, silencing of miR-23/27 suppresses l...

Age-related macular degeneration (AMD) is a degenerative disease of the retina and the leading cause of blindness in the elderly. Retinal pigment epithelial (RPE) cell death and the resultant photoreceptor apoptosis are characteristic of late-stage dry AMD, especially geographic atrophy (GA). Although oxidative stress and inflammation have been associated with GA, the nature and underlying mechanism for RPE cell death remains controversial, which hinders the development of targeted therapy for dry AMD. The purpose of this study is to systematically dissect the mechanism of RPE cell death induced by oxidative stress. Our results show that characteristic features of apoptosis, including DNA fragmentation, caspase 3 activation, chromatin condensation and apoptotic body formation, were not observed during RPE cell death induced by either hydrogen peroxide or tert-Butyl hydroperoxide. Instead, this kind of cell death can be prevented by RIP kinase inhibitors necrostatins but not caspase inhibitor z-VAD, suggesting necrotic feature of RPE cell death. Moreover, ATP depletion, receptor interacting protein kinase 3 (RIPK3) aggregation, nuclear and plasma membrane leakage and breakdown, which are the cardinal features of necrosis, were observed in RPE cells upon oxidative stress. Silencing of RIPK3, a key protein in necrosis, largely prevented oxidative stress-induced RPE death. The necrotic nature of RPE death is consistent with the release of nuclear protein high mobility group protein B1 into the cytoplasm and cell medium, which induces the expression of inflammatory gene TNFα in healthy RPE and THP-1 cells. Interestingly, features of pyroptosis or autophagy were not observed in oxidative stress-treated RPE cells. Our results unequivocally show that necrosis, but not apoptosis, is a major type of cell death in RPE cells in response to oxidative stress. This suggests that preventing oxidative stress-induced necrotic RPE death may be a viable approach for late-stage dry AMD.

Riboflavin kinase (RFK) is an essential enzyme catalyzing the phosphorylation of riboflavin (vitamin B(2)) to form FMN, an obligatory step in vitamin B(2) utilization and flavin cofactor synthesis. The structure of human RFK revealed a six-stranded antiparallel beta barrel core structurally similar to the riboflavin synthase/ferredoxin reductase FAD binding domain fold. The binding site of an intrinsically bound MgADP defines a novel nucleotide binding motif that encompasses a loop, a 3(10) helix, and a reverse turn followed by a short beta strand. This active site loop forms an arch with ATP and riboflavin binding at the opposite side and the phosphoryl transfer appears to occur through the hole underneath the arch. The invariant residues Asn36 and Glu86 are implicated in the catalysis.