SUMMARY Alu RNA accumulation due to DICER1 deficiency in the retinal pigmented epithelium (RPE) is implicated in geographic atrophy (GA), an advanced form of age-related macular degeneration that causes blindness in millions of individuals. The mechanism of Alu RNA-induced cytotoxicity is unknown. Here we show that DICER1 deficit or Alu RNA exposure activates the NLRP3 inflammasome and triggers TLR-independent MyD88 signaling via IL-18 in the RPE. Genetic or pharmacological inhibition of inflammasome components (NLRP3, Pycard, Caspase-1), MyD88, or IL-18 prevents RPE degeneration induced by DICER1 loss or Alu RNA exposure. These findings, coupled with our observation that human GA RPE contains elevated amounts of NLRP3, PYCARD and IL-18, and evidence of increased Caspase-1 and MyD88 activation, provide a rationale for targeting this pathway in GA. Our findings also reveal a function of the inflammasome outside the immune system and an immunomodulatory action of mobile elements.
Geographic atrophy (GA), an untreatable advanced form of age-related macular degeneration, results from retinal pigmented epithelium (RPE) cell death. Here we show that the microRNA (miRNA)-processing enzyme DICER1 is reduced in the RPE of humans with GA, and that conditional ablation of Dicer1, but not seven other miRNA-processing enzymes, induces RPE degeneration in mice. DICER1 knockdown induces accumulation of Alu RNA in human RPE cells and Alu-like B1 and B2 RNAs in mouse RPE. Alu RNA is increased in the RPE of humans with GA, and this pathogenic RNA induces human RPE cytotoxicity and RPE degeneration in mice. Antisense oligonucleotides targeting Alu/B1/B2 RNAs prevent DICER1 depletion-induced RPE degeneration despite global miRNA downregulation. DICER1 degrades Alu RNA, and this digested Alu RNA cannot induce RPE degeneration in mice. These findings reveal a miRNA-independent cell survival function for DICER1 involving retrotransposon transcript degradation, show that Alu RNA can directly cause human pathology, and identify new targets for a major cause of blindness.
BACKGROUND. Noninvasive detection of Alzheimer's disease (AD) with high specificity and sensitivity can greatly facilitate identification of at-risk populations for earlier, more effective intervention. AD patients exhibit a myriad of retinal pathologies, including hallmark amyloid β-protein (Aβ) deposits. METHODS.Burden, distribution, cellular layer, and structure of retinal Aβ plaques were analyzed in flat mounts and cross sections of definite AD patients and controls (n = 37). In a proof-of-concept retinal imaging trial (n = 16), amyloid probe curcumin formulation was determined and protocol was established for retinal amyloid imaging in live patients. RESULTS.Histological examination uncovered classical and neuritic-like Aβ deposits with increased retinal Aβ 42 plaques (4.7-fold; P = 0.0063) and neuronal loss (P = 0.0023) in AD patients versus matched controls. Retinal Aβ plaque mirrored brain pathology, especially in the primary visual cortex (P = 0.0097 to P = 0.0018; Pearson's r = 0.84-0.91). Retinal deposits often associated with blood vessels and occurred in hot spot peripheral regions of the superior quadrant and innermost retinal layers. Transmission electron microscopy revealed retinal Aβ assembled into protofibrils and fibrils. Moreover, the ability to image retinal amyloid deposits with solid-lipid curcumin and a modified scanning laser ophthalmoscope was demonstrated in live patients. A fully automated calculation of the retinal amyloid index (RAI), a quantitative measure of increased curcumin fluorescence, was constructed. Analysis of RAI scores showed a 2.1-fold increase in AD patients versus controls (P = 0.0031). CONCLUSION.The geometric distribution and increased burden of retinal amyloid pathology in AD, together with the feasibility to noninvasively detect discrete retinal amyloid deposits in living patients, may lead to a practical approach for large-scale AD diagnosis and monitoring.
Why some patients with seizures are successfully treated with antiepileptic drugs (AEDs) and others prove medically intractable is not known. Inadequate intraparenchymal drug concentration is a possible mechanism of resistance to AEDs. The multiple drug resistance gene (MDR1) encodes P-glycoprotein, an energy-dependent efflux pump that exports planar hydrophobic molecules from the cell. If P-glycoprotein is expressed in brain of some patients with intractable epilepsy and AEDs are exported by P-glycoprotein, lower intraparenchymal drug concentrations could contribute to lack of drug response in such patients. Eleven of 19 brain specimens removed from patients during operation for intractable epilepsy had MDR1 mRNA levels > 10 times greater than those in normal brain, as determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. Immunohistochemistry for P-glycoprotein from 14 of the patients showed increased staining in capillary endothelium in samples from epileptic patients as compared with staining in normal brain samples. In epileptic brain specimens with high MDR1 mRNA levels, expression of P-glycoprotein in astrocytes also was identified. Last, steady-state intracellular phenytoin (PHT) concentrations in MDR1 expressing neuroectodermal cells was one fourth that in MDR1-negative cells. MDR1 expression is increased in brain of some patients with medically intractable epilepsy, suggesting that the patients' lack of response to medication may be caused by inadequate accumulation of AED in brain.
In mammalian cells, endoplasmic reticulum (ER) stress has recently been shown to induce autophagy and the induction requires the unfolded protein response (UPR) signaling pathways. However, little is known whether autophagy regulates UPR pathways and how specific UPR targets might control autophagy. Here, we demonstrated that although ER stress-induced autophagy was suppressed by class III phosphatidylinositol-3 0 -kinase (PI3KC3) inhibitor 3-methyladenine (3-MA), wortmannin and knockdown of Beclin1 using small interfering RNA (siRNA), only 3-MA suppressed UPR activation. We discovered that the UPR regulator and ER chaperone GRP78/BiP is required for stress-induced autophagy. In cells in which GRP78 expression was knocked down by siRNA, despite spontaneous activation of UPR pathways and LC3 conversion, autophagosome formation induced by ER stress as well as by nutrition starvation was inhibited. GRP78 knockdown did not disrupt PI3KC3-Beclin1 association. However, electron microscopic analysis of the intracellular organelle structure reveals that the ER, a putative membrane source for generating autophagosomal double membrane, was massively expanded and disorganized in cells in which GRP78 was knocked down. ER expansion is known to be dependent on the UPR transcription factor XBP-1. Simultaneous knockdown of GRP78 and XBP-1 recovered normal levels of stress-induced autophagosome formation. Thus, these studies uncover 3-MA as an inhibitor of UPR activation and establish GRP78 as a novel obligatory component of autophagy in mammalian cells.
Frozen brain specimens from patients with multiple sclerosis (MS) and other neurologic diseases were analyzed using immunocytochemical techniques for the presence of TNF. In brain lesions in MS, and subacute sclerosing panencephalitis, TNF+ cells were demonstrated. At the lesion site in MS, TNF+ staining is associated with both astrocytes and macrophages. These observations were not made in Alzheimer's disease or normal brain tissue. The presence of TNF in MS lesions suggests a significant role for cytokines and the immune response in disease progression.
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