Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration.

Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented Abca4-/- mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of Abca4-/- mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the Abca4-/- mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.

Early Endosome Morphology in Health and Disease.

Early endosomes are organelles that receive macromolecules and solutes from the extracellular environment. The major function of early endosomes is to sort these cargos into recycling and degradative compartments of the cell. Degradation of the cargo involves maturation of early endosomes into late endosomes, which, after acquisition of hydrolytic enzymes, form lysosomes. Endosome maturation involves recruitment of specific proteins and lipids to the early endosomal membrane, which drives changes in endosome morphology. Defects in early endosome maturation are generally accompanied by alterations in morphology, such as increase in volume and/or number. Enlarged early endosomes have been observed in Alzheimer's disease and Niemann Pick Disease type C, which also exhibit defects in endocytic sorting. This article discusses the mechanisms that regulate early endosome morphology and highlights the potential importance of endosome maturation in the retinal pigment epithelium.

Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA.

During assembly of the E. coli pre-replicative complex (pre-RC), initiator DnaA oligomers are nucleated from three widely separated high-affinity DnaA recognition sites in oriC. Oligomer assembly is then guided by low-affinity DnaA recognition sites, but is also regulated by a switch-like conformational change in oriC mediated by sequential binding of two DNA bending proteins, Fis and IHF, serving as inhibitor and activator respectively. Although their recognition sites are separated by up to 90 bp, Fis represses IHF binding and weak DnaA interactions until accumulating DnaA displaces Fis from oriC. It remains unclear whether high-affinity DnaA binding plays any role in Fis repression at a distance and it is also not known whether all high-affinity DnaA recognition sites play an equivalent role in oligomer formation. To examine these issues, we developed origin-selective recombineering methods to mutate E. coli chromosomal oriC. We found that, although oligomers were assembled in the absence of any individual high-affinity DnaA binding site, loss of DnaA binding at peripheral sites eliminated Fis repression, and made binding of both Fis and IHF essential. We propose a model in which interaction of DnaA molecules at high-affinity sites regulates oriC DNA conformation.

Endoplasmic reticulum acetyltransferases Atase1 and Atase2 differentially regulate reticulophagy, macroautophagy and cellular acetyl-CoA metabolism.

Nε-lysine acetylation in the ER lumen is a recently discovered quality control mechanism that ensures proteostasis within the secretory pathway. The acetyltransferase reaction is carried out by two type-II membrane proteins, ATase1/NAT8B and ATase2/NAT8. Prior studies have shown that reducing ER acetylation can induce reticulophagy, increase ER turnover, and alleviate proteotoxic states. Here, we report the generation of Atase1-/- and Atase2-/- mice and show that these two ER-based acetyltransferases play different roles in the regulation of reticulophagy and macroautophagy. Importantly, knockout of Atase1 alone results in activation of reticulophagy and rescue of the proteotoxic state associated with Alzheimer's disease. Furthermore, loss of Atase1 or Atase2 results in widespread adaptive changes in the cell acetylome and acetyl-CoA metabolism. Overall, our study supports a divergent role of Atase1 and Atase2 in cellular biology, emphasizing ATase1 as a valid translational target for diseases characterized by toxic protein aggregation in the secretory pathway.

Centrosome Amplification in Cancer Disrupts Autophagy and Sensitizes to Autophagy Inhibition.

Centrosome amplification (CA), or a numerical increase in centrosomes, is common in human cancers, particularly those with high-risk features. We have discovered that cells with CA have an increased burden of autophagy, a catabolic process whereby autophagosomes engulf damaged organelles and proteins and deliver these contents to the lysosome for degradation and subsequent recycling. Cells with CA demonstrate an accumulation of autophagosomes. We evaluated the alternative hypotheses that CA alters autophagy by modulating microtubule networks and impairing trafficking versus altering lysosome clustering and organization versus chromosome missegregation-induced proteotoxic stress. Using LC3 reporter assays and autophagosome tracking experiments, we demonstrate that CA causes an accumulation of autophagosomes by interfering with autophagosome trafficking. To establish whether this was a druggable weakness, we tested autophagy inhibitors in our cell models of CA. Cells with CA are sensitized to chemical and genetic autophagy inhibition. Taken together, our results suggest that autophagy is disrupted by CA and sensitizes cells to inhibition of autophagy. These findings suggest a novel precision medicine strategy, whereby CA increases reliance on autophagy and serves as a biomarker for autophagy inhibitors in high-risk cancers. IMPLICATIONS: Our study suggests that CA could be used as a predictive biomarker for treatment with autophagy inhibitors.

Death following traumatic brain injury in Drosophila is associated with intestinal barrier dysfunction.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide. Unfavorable TBI outcomes result from primary mechanical injuries to the brain and ensuing secondary non-mechanical injuries that are not limited to the brain. Our genome-wide association study of Drosophila melanogaster revealed that the probability of death following TBI is associated with single nucleotide polymorphisms in genes involved in tissue barrier function and glucose homeostasis. We found that TBI causes intestinal and blood-brain barrier dysfunction and that intestinal barrier dysfunction is highly correlated with the probability of death. Furthermore, we found that ingestion of glucose after a primary injury increases the probability of death through a secondary injury mechanism that exacerbates intestinal barrier dysfunction. Our results indicate that natural variation in the probability of death following TBI is due in part to genetic differences that affect intestinal barrier dysfunction.