The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability

The evolutionary conserved TOR complex 1 controls growth in response to the quality and quantity of nutrients such as carbon and amino acids. The protein kinase Sch9 is the main TORC1 effector in yeast. However, only few of its direct targets are known. In this study, we performed a genome-wide screening looking for mutants which require Sch9 function for their survival and growth. In this way, we identified multiple components of the highly conserved vacuolar proton pump (V-ATPase) which mediates the luminal acidification of multiple biosynthetic and endocytic organelles. Besides a genetic interaction, we found Sch9 also physically interacts with the V- ATPase to regulate its assembly state in response to glucose availability and TORC1 activity. Moreover, the interaction with the V-ATPase has consequences for ageing as it allowed Sch9 to control vacuolar pH and thereby trigger either lifespan extension or lifespan shortening. Hence, our results provide insights into the signaling mechanism coupling glucose availability, TORC1 signaling, pH homeostasis and longevity. As both Sch9 and the V-ATPase are highly conserved and implicated in various pathologies, these results offer fertile ground for further research in higher eukaryotes

Evaluation of cataract formation in fish exposed to environmental radiation at Chernobyl and Fukushima

Recent studies apparently finding deleterious effects of radiation exposure on cataract formation in birds and voles living near Chernobyl represent a major challenge to current radiation protection regulations. This study conducted an integrated assessment of radiation exposure on cataractogenesis using the most advanced technologies available to assess the cataract status of lenses extracted from fish caught at both Chernobyl in Ukraine and Fukushima in Japan. It was hypothesised that these novel data would reveal positive correlations between radiation dose and early indicators of cataract formation.The structure, function and optical properties of lenses were analysed from atomic to millimetre length scales. We measured the short-range order of the lens crystallin proteins using Small Angle X-Ray Scattering (SAXS) at both the SPring-8 and DIAMOND synchrotrons, the profile of the graded refractive index generated by these proteins, the epithelial cell density and organisation and finally the focal length of each lens.The results showed no evidence of a difference between the focal length, the epithelial cell densities, the refractive indices, the interference functions and the short-range order of crystallin proteins (X-ray diffraction patterns) in lens from fish exposed to different radiation doses. It could be argued that animals in the natural environment which developed cataract would be more likely, for example, to suffer predation leading to survivor bias. But the cross-length scale study presented here, by evaluating small scale molecular and cellular changes in the lens (pre-cataract formation) significantly mitigates against this issue

Biological mechanisms underlying ionising radiation-induced cataracts

Epidemiological data correlate exposure to ionising radiation (IR) with cataract formation. In 2012, the International Commission on Radiological Protection reduced its recommended threshold for IR effects on the eye lens to 0.5 Gy and acknowledged that the biological mechanisms underlying IR-induced cataracts are largely unknown. To determine these processes, the influence of IR on the lens epithelium, cholesterol and membrane proteins was investigated. CD1, C57BL/6J and B6C3F1 mice were exposed to 0.5 - 2 Gy at 0.3 or 0.063 Gy/ min. Lens epithelium cell density was examined at 4 and/ or 12 months post-IR. Differential response to IR in these mice was observed in a strain-, dose-, dose rate-, sex-, Ptch1+/- and Ercc2+/- -dependent manner and the data indicated that low dose IR-induced cataractogenesis requires a relatively long latency period, which is consistent with epidemiological data from longitudinal studies. The plasma membranes of lens fibre cells are among the cholesterol richest membranes in the human body and cholesterol has a vital role in biophysical properties of these membranes. Analysis of IR exposed bovine eye lens membrane extracts (5 and 50 Gy) and eye lenses taken from whole body irradiated mice (0.1 and 2 Gy) showed that IR induced a significant increase in cholesterol oxides i.e. oxysterols. Unlike the isolated lens membranes, the observed enhancement of oxysterol levels in vivo was transient. Furthermore, adding α-tocopherol to isolated lens cortex membranes before exposure to IR prevented the build-up of oxysterols. The increase of oxysterols in mice eye lenses was shown to also be age-related, and was observed in the hippocampus as well. In addition, a dose-dependent increase in protein glycation was noted after IR exposure of lens membrane extracts. Collectively, these data underline that IR contributes to the cataractogenic load through compromising lens membrane integrity and lens epithelium homeostasis

Commentary: APP as a Mediator of the Synapse Pathology in Alzheimer's Disease

status: Published onlin

Cataractogenic load – a concept to study the contribution of ionizing radiation to accelerated aging in the eye lens

Ionizing radiation (IR) damages DNA and other macromolecules, including proteins and lipids. Most cell types can repair DNA damage and cycle continuously their macromolecules as a mechanism to remove defective proteins and lipids. In those cells that lack nuclei and other organelles, such as lens fiber cells and mammalian erythrocytes, IR-induced damage to macromolecules is retained because they cannot be easily replenished. Whilst the life span for an erythrocyte is several months, the life span of a human lens is decades. There is very limited turnover in lens macromolecules, therefore the aging process greatly impacts lens structure and function over its lifetime. The lens is a tissue where biomolecular longevity, lifelong retention of its components and continued growth are integral to its homeostasis. These characteristics make the lens an excellent model to study the contribution of retained macromolecular damage over time. Epidemiological data have revealed a significant association between exposure to IR, the loss of lens optical function and the formation of cataracts (cataractogenesis) later in life. Lifestyle, genetic and environmental factors all contribute to cataractogenesis due to their effect on the aging process. Cataract is an iconic age-related disease in humans. IR is a recognised cause of cataract and the occupational lens dose limit is reduced from 150 to 20 mGy / year averaged over 5 years (ICRP Publication 118). Understanding the effects of low dose IR on the lens and its role in cataractogenesis is therefore very important. So we redefine “cataractogenic load” as a term to account for the combined lifestyle, genetic and environmental processes that increase biomolecular damage to lens macromolecules. These processes weaken metabolic defenses, increase post-translational protein modifications, and alter the lipid structure and content of the lens. IR exposure is a significant insult to the lens because of free radical generation and the ensuing oxidative stress. We support the concept that damage caused by IR compounds the aging process by increasing the cataractogenic load, hereby accelerating lens aging and its loss of function

Animal source food social and behavior change communication intervention among Girinka livestock transfer beneficiaries in Rwanda: A cluster randomized evaluation

Animal source foods (ASFs), including cow’s milk, contain essential nutrients and contribute to a healthy diet, but frequency of intake is low among children in low- and middle-income countries. We hypothesized that an ASF social and behavior change communication (SBCC) intervention implemented by community health workers (CHWs) would increase child milk consumption and dietary diversity in households that received a cow from the Government of Rwanda’s Girinka livestock transfer program. We tested the 9-month SBCC intervention among children aged 12–29 months at baseline in administrative cells randomly assigned to the intervention or control. Most mothers in the intervention group were exposed to CHWs’ home visits (90.7%) or community-level activities (82.8%). At endline, more mothers in the intervention group compared with the control group knew that cow’s milk was an ASF (90.1% vs. 81.7%, P=.03) and could be introduced to children at 12 months (41.7% vs. 18.7%, P<.001). More mothers in the intervention group compared with the control group knew they should feed their children ASFs (76.2% vs. 62.1%, P=.01) and give them 1 cup of cow’s milk per day (20.6% vs. 7.8%, P<.001). Children’s consumption of fresh cow’s milk 2 or more times per week increased in the intervention group, although not significantly (8.0 percentage points, P=.17); minimum dietary diversity was unchanged. Children in the intervention group had increased odds of consuming cow’s milk 2 or more times per week if their mothers recalled hearing that children should drink 1 cup of cow’s milk per day during a CHW’s home visit [odds ratio (OR) 2.1, 95% confidence interval (CI) (1.1, 3.9)] or a community activity [OR 2.0, 95% CI (1.2, 3.5)]. Approximately half of the children had no milk during the past week because their households produced too little or sold what was produced. In poor households receiving a livestock transfer, strategies to further tailor SBCC and increase cow’s milk production may be needed to achieve larger increases in children’s frequency of milk consumption

Three-dimensional data capture and analysis of intact eye lenses evidences emmetropia-associated changes and strain-dependent differences in epithelial cell organization

Organ and tissue development are highly coordinated processes; lens growth and functional integration into the eye (emmetropia) is a robust example. An epithelial monolayer covers the anterior hemisphere of the lens, and its organization is the key to lens formation and its optical properties throughout all life stages. To better understand how the epithelium supports lens function, we have developed a novel whole tissue imaging system using conventional confocal light microscopy and a specialized analysis software to produce three-dimensional maps for the epithelium of intact mouse lenses. The open source software package geometrically determines the anterior pole position, the equatorial diameter, and three-dimensional coordinates for each detected cell in the epithelium. The user-friendly cell maps, which retain global lens geometry, allow us to document age-dependent changes in the C57/BL6J mouse lens cell distribution characteristics. We evidence changes in epithelial cell density and distribution in C57/BL6J mice during the establishment of emmetropia between postnatal weeks 4–6. These epithelial changes accompany a previously unknown spheroid to lentoid shape transition of the lens as detected by our analyses. When combined with key findings from previous mouse genetic and cell biological studies, we suggest a cytoskeleton-based mechanism likely underpins these observations

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434–451). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 was, however, not required for plasma membrane association, but biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalised with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various naturally occurring C-terminus fragments of BFSP1. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The full-length C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments enhance regulation by elevated calcium or reverse the wild type response. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations

Independent Membrane Binding Properties of the Caspase Generated Fragments of the Beaded Filament Structural Protein 1 (BFSP1) Involves an Amphipathic Helix.

BACKGROUND: BFSP1 (beaded filament structural protein 1) is a plasma membrane, Aquaporin 0 (AQP0/MIP)-associated intermediate filament protein expressed in the eye lens. BFSP1 is myristoylated, a post-translation modification that requires caspase cleavage at D433. Bioinformatic analyses suggested that the sequences 434-452 were α-helical and amphipathic. METHODS AND RESULTS: By CD spectroscopy, we show that the addition of trifluoroethanol induced a switch from an intrinsically disordered to a more α-helical conformation for the residues 434-467. Recombinantly produced BFSP1 fragments containing this amphipathic helix bind to lens lipid bilayers as determined by surface plasmon resonance (SPR). Lastly, we demonstrate by transient transfection of non-lens MCF7 cells that these same BFSP1 C-terminal sequences localise to plasma membranes and to cytoplasmic vesicles. These can be co-labelled with the vital dye, lysotracker, but other cell compartments, such as the nuclear and mitochondrial membranes, were negative. The N-terminal myristoylation of the amphipathic helix appeared not to change either the lipid affinity or membrane localisation of the BFSP1 polypeptides or fragments we assessed by SPR and transient transfection, but it did appear to enhance its helical content. CONCLUSIONS: These data support the conclusion that C-terminal sequences of human BFSP1 distal to the caspase site at G433 have independent membrane binding properties via an adjacent amphipathic helix