Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration.

Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here, we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease.

A drosophila genetic resource of mutants to study mechanisms underlying human genetic diseases.

Invertebrate model systems are powerful tools for studying human disease owing to their genetic tractability and ease of screening. We conducted a mosaic genetic screen of lethal mutations on the Drosophila X chromosome to identify genes required for the development, function, and maintenance of the nervous system. We identified 165 genes, most of whose function has not been studied in vivo. In parallel, we investigated rare variant alleles in 1,929 human exomes from families with unsolved Mendelian disease. Genes that are essential in flies and have multiple human homologs were found to be likely to be associated with human diseases. Merging the human data sets with the fly genes allowed us to identify disease-associated mutations in six families and to provide insights into microcephaly associated with brain dysgenesis. This bidirectional synergism between fly genetics and human genomics facilitates the functional annotation of evolutionarily conserved genes involved in human health.

A mitocentric view of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease, yet the underlying causative molecular mechanisms are ill defined. Numerous observations based on drug studies and mutations in genes that cause PD point to a complex set of rather subtle mitochondrial defects that may be causative. Indeed, intensive investigation of these genes in model organisms has revealed roles in the electron transport chain, mitochondrial protein homeostasis, mitophagy, and the fusion and fission of mitochondria. Here, we attempt to synthesize results from experimental studies in diverse systems to define the precise function of these PD genes, as well as their interplay with other genes that affect mitochondrial function. We propose that subtle mitochondrial defects in combination with other insults trigger the onset and progression of disease, in both familial and idiopathic PD.

Complete genome sequence of a novel secovirid infecting cassava in the Americas.

We report the complete genome sequence of a field isolate of a novel bipartite secovirid infecting cassava in Colombia, provisionally named "cassava torrado-like virus" (CsTLV). The genome sequence was obtained using Oxford Nanopore Technology, and the 5' ends were confirmed by RACE. The RNA1 is 7252 nucleotides (nt) long, encoding a polyprotein of 2336 amino acids (aa) containing the typical "replication block", conserved torradovirus motifs, and a Maf/Ham1 domain, which is not commonly found in viral genomes. The RNA2 is 4469 nt long and contains two overlapping ORFs encoding proteins of 226 and 1179 aa, showing the characteristic genome arrangement of members of the genus Torradovirus.

Correction: Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

[This corrects the article DOI: 10.1371/journal.pbio.1002197.].

The phenotype of peritoneal mouse macrophages depends on the mitochondria and ATP/ADP homeostasis.

Different macrophage subtypes have different morphologies/shapes and functions. Naïve M0 macrophages are elongated. Pro-inflammatory M1 that produce the bactericidal molecule iNos are round. Anti-inflammatory M2 macrophages that produce the pro-healing enzyme Arg-1 are highly elongated. We showed previously that the morphologies of M0 and M2 but not M1 macrophages are RhoA-dependent. Macrophage-specific deletion of RhoA causes the extreme elongation (hummingbird phenotype) of M0 and M2 but not M1 macrophages. The M1 and M2 macrophages also differ in their metabolic status. Here, we studied the effect of the oxidative phosphorylation inhibitors, antimycin A and oligomycin A, at a suboptimal dose, which depolarizes mitochondria but does not eliminate mitochondrial functions, on the mitochondria/energy production and phenotype of wild-type and RhoA-deleted M0, M1 and M2 peritoneal mouse macrophages. We found that, while untreated M1 macrophages had the lowest and the M2 had the highest level of ATP the ATP/ADP ratio was nearly identical between M0, M1 and M2 macrophages. Inhibitor treatment resulted in approximately 60% increase in ATP level and ATP/ADP ratio in M0 and M2 macrophages, and decrease in the level of filamentous (F) actin, and these changes correlated with a drastic shortening/tail retraction of M0 and M2 macrophages, and decreased expression of Arg-1 in M2 macrophages. The treatment of M1 macrophages caused only a 30% increase in the ATP level and ATP/ADP ratio, and while it did not affect the shape of M1 macrophages, it increased the production of iNos. This indicates that the maintenance of mouse macrophage phenotypes depends on mitochondrial function and ATP/ADP homeostasis.

Impaired Mitochondrial Energy Production Causes Light-Induced Photoreceptor Degeneration Independent of Oxidative Stress.

Two insults often underlie a variety of eye diseases including glaucoma, optic atrophy, and retinal degeneration--defects in mitochondrial function and aberrant Rhodopsin trafficking. Although mitochondrial defects are often associated with oxidative stress, they have not been linked to Rhodopsin trafficking. In an unbiased forward genetic screen designed to isolate mutations that cause photoreceptor degeneration, we identified mutations in a nuclear-encoded mitochondrial gene, ppr, a homolog of human LRPPRC. We found that ppr is required for protection against light-induced degeneration. Its function is essential to maintain membrane depolarization of the photoreceptors upon repetitive light exposure, and an impaired phototransduction cascade in ppr mutants results in excessive Rhodopsin1 endocytosis. Moreover, loss of ppr results in a reduction in mitochondrial RNAs, reduced electron transport chain activity, and reduced ATP levels. Oxidative stress, however, is not induced. We propose that the reduced ATP level in ppr mutants underlies the phototransduction defect, leading to increased Rhodopsin1 endocytosis during light exposure, causing photoreceptor degeneration independent of oxidative stress. This hypothesis is bolstered by characterization of two other genes isolated in the screen, pyruvate dehydrogenase and citrate synthase. Their loss also causes a light-induced degeneration, excessive Rhodopsin1 endocytosis and reduced ATP without concurrent oxidative stress, unlike many other mutations in mitochondrial genes that are associated with elevated oxidative stress and light-independent photoreceptor demise.

Correction: the retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration.

[This corrects the article DOI: 10.1371/journal.pbio.1001847.].

A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis.

Autophagy helps deliver sequestered intracellular cargo to lysosomes for proteolytic degradation and thereby maintains cellular homeostasis by preventing accumulation of toxic substances in cells. In a forward mosaic screen in Drosophila designed to identify genes required for neuronal function and maintenance, we identified multiple cacophony (cac) mutant alleles. They exhibit an age-dependent accumulation of autophagic vacuoles (AVs) in photoreceptor terminals and eventually a degeneration of the terminals and surrounding glia. cac encodes an α1 subunit of a Drosophila voltage-gated calcium channel (VGCC) that is required for synaptic vesicle fusion with the plasma membrane and neurotransmitter release. Here, we show that cac mutant photoreceptor terminals accumulate AV-lysosomal fusion intermediates, suggesting that Cac is necessary for the fusion of AVs with lysosomes, a poorly defined process. Loss of another subunit of the VGCC, α2δ or straightjacket (stj), causes phenotypes very similar to those caused by the loss of cac, indicating that the VGCC is required for AV-lysosomal fusion. The role of VGCC in AV-lysosomal fusion is evolutionarily conserved, as the loss of the mouse homologues, Cacna1a and Cacna2d2, also leads to autophagic defects in mice. Moreover, we find that CACNA1A is localized to the lysosomes and that loss of lysosomal Cacna1a in cerebellar cultured neurons leads to a failure of lysosomes to fuse with endosomes and autophagosomes. Finally, we show that the lysosomal CACNA1A but not the plasma-membrane resident CACNA1A is required for lysosomal fusion. In summary, we present a model in which the VGCC plays a role in autophagy by regulating the fusion of AVs with lysosomes through its calcium channel activity and hence functions in maintaining neuronal homeostasis.

Large-scale identification of chemically induced mutations in Drosophila melanogaster.

Forward genetic screens using chemical mutagens have been successful in defining the function of thousands of genes in eukaryotic model organisms. The main drawback of this strategy is the time-consuming identification of the molecular lesions causative of the phenotypes of interest. With whole-genome sequencing (WGS), it is now possible to sequence hundreds of strains, but determining which mutations are causative among thousands of polymorphisms remains challenging. We have sequenced 394 mutant strains, generated in a chemical mutagenesis screen, for essential genes on the Drosophila X chromosome and describe strategies to reduce the number of candidate mutations from an average of -3500 to 35 single-nucleotide variants per chromosome. By combining WGS with a rough mapping method based on large duplications, we were able to map 274 (-70%) mutations. We show that these mutations are causative, using small 80-kb duplications that rescue lethality. Hence, our findings demonstrate that combining rough mapping with WGS dramatically expands the toolkit necessary for assigning function to genes.

The retromer complex is required for rhodopsin recycling and its loss leads to photoreceptor degeneration.

Rhodopsin mistrafficking can cause photoreceptor (PR) degeneration. Upon light exposure, activated rhodopsin 1 (Rh1) in Drosophila PRs is internalized via endocytosis and degraded in lysosomes. Whether internalized Rh1 can be recycled is unknown. Here, we show that the retromer complex is expressed in PRs where it is required for recycling endocytosed Rh1 upon light stimulation. In the absence of subunits of the retromer, Rh1 is processed in the endolysosomal pathway, leading to a dramatic increase in late endosomes, lysosomes, and light-dependent PR degeneration. Reducing Rh1 endocytosis or Rh1 levels in retromer mutants alleviates PR degeneration. In addition, increasing retromer abundance suppresses degenerative phenotypes of mutations that affect the endolysosomal system. Finally, expressing human Vps26 suppresses PR degeneration in Vps26 mutant PRs. We propose that the retromer plays a conserved role in recycling rhodopsins to maintain PR function and integrity.

Drosophila Tempura, a novel protein prenyltransferase α subunit, regulates notch signaling via Rab1 and Rab11.

Vesicular trafficking plays a key role in tuning the activity of Notch signaling. Here, we describe a novel and conserved Rab geranylgeranyltransferase (RabGGT)-α-like subunit that is required for Notch signaling-mediated lateral inhibition and cell fate determination of external sensory organs. This protein is encoded by tempura, and its loss affects the secretion of Scabrous and Delta, two proteins required for proper Notch signaling. We show that Tempura forms a heretofore uncharacterized RabGGT complex that geranylgeranylates Rab1 and Rab11. This geranylgeranylation is required for their proper subcellular localization. A partial dysfunction of Rab1 affects Scabrous and Delta in the secretory pathway. In addition, a partial loss Rab11 affects trafficking of Delta. In summary, Tempura functions as a new geranylgeranyltransferase that regulates the subcellular localization of Rab1 and Rab11, which in turn regulate trafficking of Scabrous and Delta, thereby affecting Notch signaling.

Crag is a GEF for Rab11 required for rhodopsin trafficking and maintenance of adult photoreceptor cells.

Rhodopsins (Rhs) are light sensors, and Rh1 is the major Rh in the Drosophila photoreceptor rhabdomere membrane. Upon photoactivation, a fraction of Rh1 is internalized and degraded, but it remains unclear how the rhabdomeric Rh1 pool is replenished and what molecular players are involved. Here, we show that Crag, a DENN protein, is a guanine nucleotide exchange factor for Rab11 that is required for the homeostasis of Rh1 upon light exposure. The absence of Crag causes a light-induced accumulation of cytoplasmic Rh1, and loss of Crag or Rab11 leads to a similar photoreceptor degeneration in adult flies. Furthermore, the defects associated with loss of Crag can be partially rescued with a constitutive active form of Rab11. We propose that upon light stimulation, Crag is required for trafficking of Rh from the trans-Golgi network to rhabdomere membranes via a Rab11-dependent vesicular transport.

Essential role for Nix in autophagic maturation of erythroid cells.

Erythroid cells undergo enucleation and the removal of organelles during terminal differentiation. Although autophagy has been suggested to mediate the elimination of organelles for erythroid maturation, the molecular mechanisms underlying this process remain undefined. Here we report a role for a Bcl-2 family member, Nix (also called Bnip3L), in the regulation of erythroid maturation through mitochondrial autophagy. Nix(-/-) mice developed anaemia with reduced mature erythrocytes and compensatory expansion of erythroid precursors. Erythrocytes in the peripheral blood of Nix(-/-) mice exhibited mitochondrial retention and reduced lifespan in vivo. Although the clearance of ribosomes proceeded normally in the absence of Nix, the entry of mitochondria into autophagosomes for clearance was defective. Deficiency in Nix inhibited the loss of mitochondrial membrane potential (DeltaPsi(m)), and treatment with uncoupling chemicals or a BH3 mimetic induced the loss of DeltaPsi(m) and restored the sequestration of mitochondria into autophagosomes in Nix(-/-) erythroid cells. These results suggest that Nix-dependent loss of DeltaPsi(m) is important for targeting the mitochondria into autophagosomes for clearance during erythroid maturation, and interference with this function impairs erythroid maturation and results in anaemia. Our study may also provide insights into molecular mechanisms underlying mitochondrial quality control involving mitochondrial autophagy.

Surgical outcomes of intraocular lens iris suture fixation in eyes with residual capsule support.

PURPOSE: To evaluate the safety and refractive outcomes of eyes after intraocular lens (IOL) iris suture fixation (ISF). SETTING: Private practice, Los Angeles, California. DESIGN: Nonrandomized and unmasked retrospective chart review. METHODS: Eyes that underwent IOL exchange or repositioning with ISF with at least 270 degrees of capsular support were included. Eyes with less than 270 degrees of capsular support and eyes with iris damage were excluded. The primary outcome measures included incidence of cystoid macular edema (CME), IOL dislocation requiring refixation, and chronic inflammation. Secondary outcome measures included worsening intraocular pressure (IOP) control, retinal tear or detachment, worsening of corrected distance visual acuity (CDVA), and corneal decompensation. Refractive outcomes for 26 subgrouped eyes included mean and median spherical equivalent refraction accuracy (SERA), and percentage of eyes within 0.5 diopter (D) and 1 D of the refractive target. RESULTS: The study included 53 eyes of 50 patients. CME: 2/53 (3.8%), IOL dislocation requiring refixation: 2/53 (3.8%), chronic inflammation: 1/53 (1.9%), worsening IOP control: 5/53 (9.4%), retinal tear or detachment: 2/53 (3.8%). No patient experienced worsening of CDVA from baseline or corneal decompensation. Mean SERA ± SD -0.35 ± 0.29 D, median SERA -0.37 D. Of the 26 eyes subgrouped for refractive analysis, 73% were within 0.5 D and 100% were within 1 D of the desired refractive outcome. CONCLUSIONS: ISF can offer stability for sulcus-fixated IOLs provided there is some residual capsule support. Although there are measurable complications, there is a relatively low side effect profile. The refractive error tended to be myopic, indicating the need for further refinement of IOL power predictive formulas.

Clinical outcomes and complications following intraocular lens exchange in the setting of an open or intact posterior capsule.

PURPOSE: To assess whether there are added risks when performing intraocular lens (IOL) exchange in the setting of an open posterior capsule (OPC) when compared with a closed posterior capsule (CPC) IOL exchange. SETTING: Private practice, Los Angeles, California. DESIGN: Nonrandomized and unmasked retrospective chart review. METHODS: Eyes undergoing IOL exchange solely to relieve optical symptoms, with open or intact posterior capsules, were included. Eyes undergoing IOL exchange due to IOL malposition or dislocation were excluded. Eyes with preexisting, uncontrolled glaucoma and inflammation and eyes with a visual potential worse than 20/40 (Snellen) were also excluded. The main outcome measures were the postoperative complications compared between the OPC and CPC groups. RESULTS: 90 eyes of 75 patients undergoing IOL exchange were included in this study; 38/90 eyes had an OPC, and 52/90 eyes had a CPC. 3/38 in the OPC group and 2/52 in the CPC group experienced worsening intraocular pressure control. 1/38 in the OPC group experienced chronic inflammation. 2/38 in the OPC group and 2/52 in the CPC group experienced cystoid macular edema. 1/52 in the CPC group experienced a retinal tear. Statistically or clinically significant differences in postoperative complications between the OPC and CPC groups were not found. CONCLUSIONS: In the hands of an experienced surgeon, IOL exchange with an OPC appear red to be just as safe as IOL exchange with a CPC; when deemed necessary, experienced surgeons may perform an IOL exchange safely in the presence of an OPC.

Amino acid residues in Rag1 crucial for DNA hairpin formation.

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.

Regulation of B cell fate, survival, and function by mitochondria and autophagy.

B cells are responsible for protective antibody production after differentiation into antibody-secreting cells during humoral immune responses. From early B cell development in the bone marrow, to their maturation in the periphery, activation in the germinal center, and differentiation into plasma cells or memory B cells, B cells display ever-changing functions and properties. Autophagy and mitochondria play important roles in B cell development, activation, and differentiation to accommodate the phenotypic and environmental changes encountered over the lifetime of the cell. Among their many functions, mitochondria and autophagy generate energy, mediate cell survival, and produce/eliminate reactive oxygen species that can serve as signal molecules to regulate differentiation. As B cells mature and differentiate into plasma or memory cells, both autophagic and mitochondrial functions undergo significant changes. In this review, we aim to provide an overview of the role of the autophagosome and mitochondria in regulating B cell fate, survival, and function. Moreover, we will discuss the interplay between these two highly metabolic organelles during B cell development, maturation, and differentiation.

[Survival of Gastric Cancer in Western Honduras Pilot study: 2002-2012]. / SUPERVIVENCIA DE CÁNCER GÁSTRICO EN EL OCCIDENTE DE HONDURAS ESTUDIO PILOTO: 2002­2012.

Background: Gastric cancer is the second leading cause of cancer death globally. In Honduras the incidence in the last decade was 39 and 21 per 100,000 inhabitants for men and women, respectively. In 2008 IARC (GLOBOCAN) placed Honduras as the country with the highest incidence of gastric cancer in Latin America. Methods: A retrospective cohort study of patients diagnosed with gastric cancer at the Hospital de Occidente between 2002-2012 was designed. A sample of 144 patients was selected from a total of 490 to obtain a confidence level of 95%. The data collection was obtained by verbal autopsy. Prognostic factors of survival were analyzed using Cox proportional hazards ratio models (CI: 95%). Outcomes: The male/female ratio was 2.8:1. The mean age was 63.29 years. Overall five-year survival was 9.39%. Among patients receiving dual therapy (surgery and chemotherapy), a statistically significant increase in survival was found (10.42%, p=0.048). Between the proximal (28.95%) and distal (56.58%) locations also a statistically significant difference was observed (p=0.03). There was no statistically significant difference in the macroscopic (Borrmann) and microscopic findings (Lauren). Disscusion: This study represents the first effort to estimate survival of gastric cancer in Honduras. Survival may be linked to the location of the primary lesion and the type of treatment. It is expected to develop studies with greater coverage, to answer these questions.