Lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) lncRNA differentially regulates gene and protein expression, signaling and morphology of human ocular cells.

Pseudoexfoliation glaucoma (PEXG) is characterized by dysregulated extracellular matrix (ECM) homeostasis that disrupts conventional outflow function and increases intraocular pressure (IOP). Prolonged IOP elevation results in optic nerve head damage and vision loss. Uniquely, PEXG is a form of open angle glaucoma that has variable penetrance, is difficult to treat and does not respond well to common IOP-lowering pharmaceuticals. Therefore, understanding modulators of disease severity will aid in targeted therapies for PEXG. Genome-wide association studies have identified polymorphisms in the long non-coding RNA lysyl oxidase-like 1-antisense 1 (LOXL1-AS1) as a risk factor for PEXG. Risk alleles, oxidative stress and mechanical stretch all alter LOXL1-AS1 expression. As a long non-coding RNA, LOXL1-AS1 binds hnRNPL and regulates global gene expression. In this study, we focus on the role of LOXL1-AS1 in the ocular cells (trabecular meshwork and Schlemm's canal) that regulate IOP. We show that selective knockdown of LOXL1-AS1 leads to cell-type-specific changes in gene expression, ECM homeostasis, signaling and morphology. These results implicate LOXL1-AS1 as a modulator of cellular homeostasis, altering cell contractility and ECM turnover, both of which are well-known contributors to PEXG. These findings support LOXL1-AS1 as a key target for modifying the disease.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

EXTRA SPINDLE POLES (Separase) controls anisotropic cell expansion in Norway spruce (Picea abies) embryos independently of its role in anaphase progression.

The caspase-related protease separase (EXTRA SPINDLE POLES, ESP) plays a major role in chromatid disjunction and cell expansion in Arabidopsis thaliana. Whether the expansion phenotypes are linked to defects in cell division in Arabidopsis ESP mutants remains elusive. Here we present the identification, cloning and characterization of the gymnosperm Norway spruce (Picea abies, Pa) ESP. We used the P. abies somatic embryo system and a combination of reverse genetics and microscopy to explore the roles of Pa ESP during embryogenesis. Pa ESP was expressed in the proliferating embryonal mass, while it was absent in the suspensor cells. Pa ESP associated with kinetochore microtubules in metaphase and then with anaphase spindle midzone. During cytokinesis, it localized on the phragmoplast microtubules and on the cell plate. Pa ESP deficiency perturbed anisotropic expansion and reduced mitotic divisions in cotyledonary embryos. Furthermore, whilst Pa ESP can rescue the chromatid nondisjunction phenotype of Arabidopsis ESP mutants, it cannot rescue anisotropic cell expansion. Our data demonstrate that the roles of ESP in daughter chromatid separation and cell expansion are conserved between gymnosperms and angiosperms. However, the mechanisms of ESP-mediated regulation of cell expansion seem to be lineage-specific.

Matrix metalloproteinase Mmp-1a is dispensable for normal growth and fertility in mice and promotes lung cancer progression by modulating inflammatory responses.

Human MMP-1 is a matrix metalloproteinase repeatedly associated with many pathological conditions, including cancer. Thus, MMP1 overexpression is a poor prognosis marker in a variety of advanced cancers, including colorectal, breast, and lung carcinomas. Moreover, MMP-1 plays a key role in the metastatic behavior of melanoma, breast, and prostate cancer cells. However, functional and mechanistic studies on the relevance of MMP-1 in cancer have been hampered by the absence of an in vivo model. In this work, we have generated mice deficient in Mmp1a, the murine ortholog of human MMP1. Mmp1a(-/-) mice are viable and fertile and do not exhibit obvious abnormalities, which has facilitated studies of cancer susceptibility. These studies have shown a decreased susceptibility to develop lung carcinomas induced by chemical carcinogens in Mmp1a(-/-) mice. Histopathological analysis indicated that tumors generated in Mmp1a(-/-) mice are smaller than those of wild-type mice, consistently with the idea that the absence of Mmp-1a hampers tumor progression. Proteomic analysis revealed decreased levels of chitinase-3-like 3 and accumulation of the receptor for advanced glycation end-products and its ligand S100A8 in lung samples from Mmp1a(-/-) mice compared with those from wild-type. These findings suggest that Mmp-1a could play a role in tumor progression by modulating the polarization of a Th1/Th2 inflammatory response to chemical carcinogens. On the basis of these results, we propose that Mmp1a knock-out mice provide an excellent in vivo model for the functional analysis of human MMP-1 in both physiological and pathological conditions.

ADAMTS-12 metalloprotease is necessary for normal inflammatory response.

ADAMTSs (a disintegrin and metalloprotease with thrombospondin domains) are a family of enzymes with both proteolytic and protein interaction functions, which have been implicated in distinct pathologies. In this work, we have investigated the putative role of ADAMTS-12 in inflammation by using a mouse model deficient in this metalloprotease. Control and mutant mice were subjected to different experimental conditions to induce colitis, endotoxic sepsis, and pancreatitis. We have observed that Adamts12-deficient mice exhibit more severe inflammation and a delayed recovery from these challenges compared with their wild-type littermates. These changes are accompanied by an increase in inflammatory markers including several cytokines, as assessed by microarray expression analysis and proteomic-based approaches. Interestingly, the clinical symptoms observed in Adamts12-deficient mice are also concomitant with an elevation in the number of neutrophils in affected tissues. Finally, isolation and in vitro culture of human neutrophils demonstrate that the presence of ADAMTS-12 induces neutrophil apoptosis. On the basis of these results, we propose that ADAMTS-12 is implicated in the inflammatory response by modulating normal neutrophil apoptosis.

Analysis of ATG4C function in vivo.

ABBREVIATIONS: ATG4 (autophagy related 4 cysteine peptidase); ATG4A (autophagy related 4A cysteine peptidase); ATG4B (autophagy related 4B cysteine peptidase); ATG4C (autophagy related 4C cysteine peptidase); ATG4D (autophagy related 4D cysteine peptidase); Atg8 (autophagy related 8); GABARAP (GABA type A receptor-associated protein); GABARAPL1(GABA type A receptor-associated protein like 1); GABARAPL2 (GABA type A receptor-associated protein like 2); MAP1LC3A/LC3A (microtubule associated protein 1 light chain 3 alpha); MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta); mATG8 (mammalian Atg8); PE (phosphatidylethanolamine); PS (phosphatydylserine); SQSTM1/p62 (sequestosome 1).

Genetic background determines severity of Loxl1-mediated systemic and ocular elastosis in mice.

Pseudoexfoliation syndrome (PEX) is a systemic, age-related disorder characterized by elastosis and extracellular matrix deposits. Its most significant ocular manifestation is an aggressive form of glaucoma associated with variants in the gene encoding lysyl oxidase-like 1 (LOXL1). Depending upon the population, variants in LOXL1 can impart risk or protection for PEX, suggesting the importance of genetic context. As LOXL1 protein levels are lower and the degree of elastosis is higher in people with PEX, we studied Loxl1-deficient mice on three different genetic backgrounds: C57BL/6 (BL/6), 129S×C57BL/6 (50/50) and 129S. Early onset and high prevalence of spontaneous pelvic organ prolapse in BL/6 Loxl1-/- mice necessitated the study of mice that were <2 months old. Similar to pelvic organ prolapse, most elastosis endpoints were the most severe in BL/6 Loxl1-/- mice, including skin laxity, pulmonary tropoelastin accumulation, expansion of Schlemm's canal and dilation of intrascleral veins. Interestingly, intraocular pressure was elevated in 50/50 Loxl1-/- mice, depressed in BL/6 Loxl1-/- mice and unchanged in 129S Loxl1-/- mice compared to that of control littermates. Overall, the 129S background was protective against most elastosis phenotypes studied. Thus, repair of elastin-containing tissues is impacted by the abundance of LOXL1 and genetic context in young animals.

ATG4D is the main ATG8 delipidating enzyme in mammalian cells and protects against cerebellar neurodegeneration.

Despite the great advances in autophagy research in the last years, the specific functions of the four mammalian Atg4 proteases (ATG4A-D) remain unclear. In yeast, Atg4 mediates both Atg8 proteolytic activation, and its delipidation. However, it is not clear how these two roles are distributed along the members of the ATG4 family of proteases. We show that these two functions are preferentially carried out by distinct ATG4 proteases, being ATG4D the main delipidating enzyme. In mammalian cells, ATG4D loss results in accumulation of membrane-bound forms of mATG8s, increased cellular autophagosome number and reduced autophagosome average size. In mice, ATG4D loss leads to cerebellar neurodegeneration and impaired motor coordination caused by alterations in trafficking/clustering of GABAA receptors. We also show that human gene variants of ATG4D associated with neurodegeneration are not able to fully restore ATG4D deficiency, highlighting the neuroprotective role of ATG4D in mammals.

Detection of programmed cell death in plant embryos.

Programmed cell death (PCD) is an integral part of embryogenesis. In plant embryos, PCD functions during terminal differentiation and elimination of the temporary organ, suspensor, as well as during establishment of provascular system. Embryo abortion is another example of embryonic PCD activated at pathological situations and in polyembryonic seeds. Recent studies identified the sequence of cytological events leading to cellular self-destruction in plant embryos. As in most if not all the developmental cell deaths in plants, embryonic PCD is hallmarked by autophagic degradation of the cytoplasm and nuclear disassembly that includes breakdown of the nuclear envelope and DNA fragmentation. The optimized setup of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) allows the routine in situ analysis of nuclear DNA fragmentation in plant embryos. This chapter provides step-by-step procedure of how to process embryos for TUNEL and how to combine TUNEL with immunolocalization of the protein of interest.

Autophagy couteracts weight gain, lipotoxicity and pancreatic ß-cell death upon hypercaloric pro-diabetic regimens.

In the last years, autophagy has been revealed as an essential pathway for multiple biological processes and physiological functions. As a catabolic route, autophagy regulation by nutrient availability has been evolutionarily conserved from yeast to mammals. On one hand, autophagy induction by starvation is associated with a significant loss in body weight in mice. Here, we demonstrate that both genetic and pharmacological inhibition of the autophagy process compromise weight loss induced by starvation. Moreover, autophagic potential also impacts on weight gain induced by distinct hypercaloric regimens. Atg4b-deficient mice, which show limited autophagic competence, exhibit a major increase in body weight in response to distinct obesity-associated metabolic challenges. This response is characterized by the presence of larger adipocytes in visceral fat tissue, increased hepatic steatosis, as well as reduced glucose tolerance and attenuated insulin responses. Similarly, autophagy-deficient mice are more vulnerable to experimentally induced type-I diabetes, showing an increased susceptibility to acute streptozotocin administration. Notably, pharmacological stimulation of autophagy in wild-type mice by spermidine reduced both weight gain and obesity-associated alterations upon hypercaloric regimens. Altogether, these results indicate that systemic autophagic activity influences the resilience of the organism to weight gain induced by high-calorie diets, as well as to the obesity-associated features of both type-1 and type-2 diabetes.

Climatic droplet keratopathy: an old disease in new clothes.

Climatic droplet keratopathy (CDK) is an acquired and potentially handicapping cornea degenerative disease that is highly prevalent in certain rural communities around the world. It predominantly affects males over their forties. It has many other names such as Bietti's band-shaped nodular dystrophy, Labrador keratopathy, spheroidal degeneration, chronic actinic keratopathy, oil droplet degeneration, elastoid degeneration and keratinoid corneal degeneration. CDK is characterized by the haziness and opalescence of the cornea's most anterior layers which go through three stages with increasing severity. Globular deposits of different sizes may be histopathologically observed under the corneal epithelium by means of light and electron microscopy. The coalescence and increased volume of these spherules may cause the disruption of Bowman's membrane and the elevation and thinning of the corneal epithelium. The exact aetiology and pathogenesis of CDK are unknown, but they are possibly multifactorial. The only treatment in CDK advanced cases is a corneal transplantation, which in different impoverished regions of the world is not an available option. Many years ago, the clinical and histological aspects of this disease were described in several articles. This review highlights new scientific evidence of the expanding knowledge on CDK's pathogenesis which will open the prospect for new therapeutic interventions.

Comparing nutrient intake from food to the estimated average requirements shows middle- to upper-income pregnant women lack iron and possibly magnesium.

OBJECTIVE: To determine whether nutrient intake from food alone was adequate across trimesters for middle- to upper-income pregnant women when compared with estimated average requirements (EAR), and to determine whether food intake exceeded the tolerable upper intake level (UL) for any nutrient. DESIGN: Observational study in which pregnant women completed 3-day diet records each month during their pregnancy. Records were analyzed for nutrient content, and usual intake distributions were determined. SUBJECTS/SETTING: Subjects were low-risk women in their first trimester of pregnancy (living in middle- to upper-income households). Ninety-four women were recruited, and sixty-three participated. STATISTICAL ANALYSIS PERFORMED: Nutrient intake data were adjusted to achieve normality by using a power transformation. A mixed model method was used to assess trends in intake over time, and to estimate mean intake and within-subjects and between-subjects variance. The usual intake distribution for each nutrient was determined and compared with the EAR and UL. RESULTS: The probabilities of usual nutrient intake from food being less than the EAR were highest for iron (.91), magnesium (.53), zinc (.31), vitamin B6 (.21), selenium (.20), and vitamin C (.12). Women were not at risk of exceeding the UL from food intake for any nutrient studied. APPLICATIONS/CONCLUSIONS: Study participants did not consume adequate amounts of iron from food to meet the needs of pregnancy, and therefore iron supplementation is warranted in this population. Intake of magnesium was suboptimal using the EAR as a cut-point for adequacy.

Variation in transcript abundance during somatic embryogenesis in gymnosperms.

Somatic embryogenesis of Norway spruce (Picea abies L.) is a versatile model system to study molecular mechanisms regulating embryo development because it proceeds through defined developmental stages corresponding to specific culture treatments. Normal embryonic development involves early differentiation of proembryogenic masses (PEMs) into somatic embryos, followed by early and late embryogeny leading to the formation of mature cotyledonary embryos. In some cell lines there is a developmental arrest at the PEM-somatic embryo transition. To learn more about the molecular mechanisms regulating embryogenesis, we compared the transcript profiles of two normal lines and one developmentally arrested line. Ribonucleic acid, extracted from these cell lines at successive developmental stages, was analyzed on DNA microarrays containing 2178 expressed sequence tags (ESTs) (corresponding to 2110 unique cDNAs) from loblolly pine (Pinus taeda L.). Hybridization between spruce and pine species on microarrays has been shown to be effective (van Zyl et al. 2002, Stasolla et al. 2003). In contrast to the developmentally arrested line, the early phases of normal embryo development are characterized by a precise pattern of gene expression, i.e., repression followed by induction. Comparison of transcript levels between successive stages of embryogenesis allowed us to identify several genes that showed unique expression responses during normal development. Several of these genes encode proteins involved in detoxification processes, methionine synthesis and utilization, and carbohydrate metabolism. The potential role of these genes in embryo development is discussed.

Autophagy mediates caloric restriction-induced lifespan extension in Arabidopsis.

Caloric restriction (CR) extends lifespan in various heterotrophic organisms ranging from yeasts to mammals, but whether a similar phenomenon occurs in plants remains unknown. Plants are autotrophs and use their photosynthetic machinery to convert light energy into the chemical energy of glucose and other organic compounds. As the rate of photosynthesis is proportional to the level of photosynthetically active radiation, the CR in plants can be modeled by lowering light intensity. Here, we report that low light intensity extends the lifespan in Arabidopsis through the mechanisms triggering autophagy, the major catabolic process that recycles damaged and potentially harmful cellular material. Knockout of autophagy-related genes results in the short lifespan and suppression of the lifespan-extending effect of the CR. Our data demonstrate that the autophagy-dependent mechanism of CR-induced lifespan extension is conserved between autotrophs and heterotrophs.