Maturation of autophagosomes and endosomes: A key role for Rab7

AbstractMacroautophagy is an important route in cellular maintenance, in the breakdown and reuse of intracellular materials. It is closely related to endocytosis, the means by which the cell can absorb extracellular material, as both macroautophagy and endocytosis have converging steps and common participating molecules. The point where autophagosomes and endosomes fuse with lysosomes to permit for the final degradation of their contents is important. One of the most substantial molecules in the maturation of autophagosomes/endosomes is Rab7, a member of small GTPases. Rab7 designates the maturation of endosomes and also autophagosomes, directing the trafficking of cargos along microtubules, and finally, participating in the fusion step with lysosomes. Rab7 is an effective multifunctional regulator of autophagy and endocytosis. Since many aggregation-based diseases, e.g. age-related macular degeneration of the eye (AMD) and Alzheimer's disease are due of malfunctioning in the autophagic process, the management of Rab7 activity might hold potential as a therapeutic target against these diseases

ER stress in Alzheimer's disease: a novel neuronal trigger for inflammation and Alzheimer's pathology

The endoplasmic reticulum (ER) is involved in several crucial cellular functions, e.g. protein folding and quality control, maintenance of Ca2+ balance, and cholesterol synthesis. Many genetic and environmental insults can disturb the function of ER and induce ER stress. ER contains three branches of stress sensors, i.e. IRE1, PERK and ATF6 transducers, which recognize the misfolding of proteins in ER and activate a complex signaling network to generate the unfolded protein response (UPR). Alzheimer's disease (AD) is a progressive neurodegenerative disorder involving misfolding and aggregation of proteins in conjunction with prolonged cellular stress, e.g. in redox regulation and Ca2+ homeostasis. Emerging evidence indicates that the UPR is activated in neurons but not in glial cells in AD brains. Neurons display pPERK, peIF2α and pIRE1α immunostaining along with abundant diffuse staining of phosphorylated tau protein. Recent studies have demonstrated that ER stress can also induce an inflammatory response via different UPR transducers. The most potent pathways are IRE1-TRAF2, PERK-eIF2α, PERK-GSK-3, ATF6-CREBH, as well as inflammatory caspase-induced signaling pathways. We will describe the mechanisms which could link the ER stress of neurons to the activation of the inflammatory response and the evolution of pathological changes in AD

A refined in vitro model to study inflammatory responses in organotypic membrane culture of postnatal rat hippocampal slices

BACKGROUND: Propagated tissue degeneration, especially during aging, has been shown to be enhanced through potentiation of innate immune responses. Neurodegenerative diseases and a wide variety of inflammatory conditions are linked together and several anti-inflammatory compounds considered as having therapeutic potential for example in Alzheimer's disease (AD). In vitro brain slice techniques have been widely used to unravel the complexity of neuroinflammation, but rarely, has the power of the model itself been reported. Our aim was to gain a more detailed insight and understanding of the behaviour of hippocampus tissue slices in serum-free, interface culture per se and after exposure to different pro- and anti-inflammatory compounds. METHODS: The responses of the slices to pro- and anti-inflammatory stimuli were monitored at various time points by measuring the leakage of lactate dehydrogenase (LDH) and the release of cytokines interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) and nitric oxide (NO) from the culture media. Histological methods were applied to reveal the morphological status after exposure to stimuli and during the time course of the culture period. Statistical power analysis were made with nQuery Advisor(®), version 5.0, (Statistical Solutions, Saugus, MA) computer program for Wilcoxon (Mann-Whitney) rank-sum test. RESULTS: By using the interface membrane culture technique, the hippocampal slices largely recover from the trauma caused by cutting after 4–5 days in vitro. Furthermore, the cultures remain stable and retain their responsiveness to inflammatory stimuli for at least 3 weeks. During this time period, cultures are susceptible to modification by inflammatory stimuli as assessed by quantitative biochemical assays and morphological characterizations. CONCLUSION: The present report outlines the techniques for studying immune responses using a serum-free slice culture model. Statistically powerful data under controlled culture conditions and with ethically justified use of animals can be obtained as soon as after 4–5 DIV. The model is most probably suitable also for studies of chronic inflammation

How does the circular economy discourse affect policy-making? The case of streamlining waste utilisation in Finnish earthworks

Since entering the waste policy debate in the 1980s, the sustainability discourse has sought to find alternatives to end-of-pipe solutions. The latest development on this path is the emergence of the circular economy, which aims to close the loop of the current linear economy. This case study analyses a substantial Finnish waste policy reform that has been underway since the late 1990s. The objective of the reform has been to create a decree that streamlines waste utilisation in earthworks. The decree was prepared between 2000 and 2006, and then reformed between 2015 and 2018. We analysed the discursive spaces of both phases and compared them to interpret the changes in the discourse of waste policy. The discursive space in the preparation phase was structured by the tension between the discourses of resource efficiency and precaution, but in the reform, the emergence of the circular economy diversified the discursive space. The thinking regarding the circular economy has added complexity, competition, and struggle to waste policy, but also enhanced the role of upcycling

Influence of Hsp90 and HDAC Inhibition and Tubulin Acetylation on Perinuclear Protein Aggregation in Human Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells are continually exposed to oxidative stress that contributes to protein misfolding, aggregation and functional abnormalities during aging. The protein aggregates formed at the cell periphery are delivered along the microtubulus network by dynein-dependent retrograde trafficking to a juxtanuclear location. We demonstrate that Hsp90 inhibition by geldanamycin can effectively suppress proteasome inhibitor, MG-132-induced protein aggregation in a way that is independent of HDAC inhibition or the tubulin acetylation levels in ARPE-19 cells. However, the tubulin acetylation and polymerization state affects the localization of the proteasome-inhibitor-induced aggregation. These findings open new perspectives for understanding the pathogenesis of protein aggregation in retinal cells and can be useful for the development of therapeutic treatments to prevent retinal cell deterioration

Hsp70 binds reversibly to proteasome inhibitor-induced protein aggregates and evades autophagic clearance in ARPE-19 cells

Age-related macular degeneration (AMD) is characterized primarily by degeneration of the macular retinal pigment epithelium (RPE) that secondarily leads to cell death of photoreceptors and impaired central vision. Hallmarks of AMD are accumulation of lysosomal lipofuscin and extracellular drusen, which indicate impaired proteolysis in RPE cells. Cellular proteostasis is strongly regulated by molecular chaperones such as Hsp70 and proteasomal and autophagic clearance systems. We have recently shown that autophagy receptor SQSTM1/p62 binds irreversibly to proteasome inhibitor–induced perinuclear protein aggregates and undergoes autophagic clearance in RPE cell cultures. Revealing decreased autophagy, SQSTM1/p62 accumulates in macular area of donor AMD patient samples. In this study, we show that Hsp70 binds reversibly to proteasome inhibitor–induced perinuclear protein aggregates and does not become degraded by autophagy in ARPE-19 cells. Our observation reveals new opportunities to use a cytoprotective Hsp70 as a therapy target in the prevention of RPE cell degeneration and development of AMD

Red clover (Trifolium pratense L.): Identification and characterization of phenolic compounds and protective action against oxidative stress and inflammation

Red clover (Trifolium pratense L.), a widely used feed crop, has recently received considerable interest as a valuable source of phenolic compounds with multiple potential protective functions. It is a rich source of isoflavonoids, plant secondary metabolites widely distributed in the Leguminosae family