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.

Buffered EGFR signaling regulated by spitz-to-argos expression ratio is a critical factor for patterning the Drosophila eye.

The Epidermal Growth Factor Receptor (EGFR) signaling pathway plays a critical role in regulating tissue patterning. Drosophila EGFR signaling achieves specificity through multiple ligands and feedback loops to finetune signaling outcomes spatiotemporally. The principal Drosophila EGF ligand, cleaved Spitz, and the negative feedback regulator, Argos are diffusible and can act both in a cell autonomous and non-autonomous manner. The expression dose of Spitz and Argos early in photoreceptor cell fate determination has been shown to be critical in patterning the Drosophila eye, but the exact identity of the cells expressing these genes in the larval eye disc has been elusive. Using single molecule RNA Fluorescence in situ Hybridization (smFISH), we reveal an intriguing differential expression of spitz and argos mRNA in the Drosophila third instar eye imaginal disc indicative of directional non-autonomous EGFR signaling. By genetically tuning EGFR signaling, we show that rather than absolute levels of expression, the ratio of expression of spitz-to-argos to be a critical determinant of the final adult eye phenotype. Proximate effects on EGFR signaling in terms of cell cycle and differentiation markers are affected differently in the different perturbations. Proper ommatidial patterning is robust to thresholds around a tightly maintained wildtype spitz-to-argos ratio, and breaks down beyond. This provides a powerful instance of developmental buffering against gene expression fluctuations.

Bendless is essential for PINK1-Park mediated Mitofusin degradation under mitochondrial stress caused by loss of LRPPRC.

Cells under mitochondrial stress often co-opt mechanisms to maintain energy homeostasis, mitochondrial quality control and cell survival. A mechanistic understanding of such responses is crucial for further insight into mitochondrial biology and diseases. Through an unbiased genetic screen in Drosophila, we identify that mutations in lrpprc2, a homolog of the human LRPPRC gene that is linked to the French-Canadian Leigh syndrome, result in PINK1-Park activation. While the PINK1-Park pathway is well known to induce mitophagy, we show that PINK1-Park regulates mitochondrial dynamics by inducing the degradation of the mitochondrial fusion protein Mitofusin/Marf in lrpprc2 mutants. In our genetic screen, we also discover that Bendless, a K63-linked E2 conjugase, is a regulator of Marf, as loss of bendless results in increased Marf levels. We show that Bendless is required for PINK1 stability, and subsequently for PINK1-Park mediated Marf degradation under physiological conditions, and in response to mitochondrial stress as seen in lrpprc2. Additionally, we show that loss of bendless in lrpprc2 mutant eyes results in photoreceptor degeneration, indicating a neuroprotective role for Bendless-PINK1-Park mediated Marf degradation. Based on our observations, we propose that certain forms of mitochondrial stress activate Bendless-PINK1-Park to limit mitochondrial fusion, which is a cell-protective response.

Photothermal modulation of human stem cells using light-responsive 2D nanomaterials.

Two-dimensional (2D) molybdenum disulfide (MoS2) nanomaterials are an emerging class of biomaterials that are photoresponsive at near-infrared wavelengths (NIR). Here, we demonstrate the ability of 2D MoS2 to modulate cellular functions of human stem cells through photothermal mechanisms. The interaction of MoS2 and NIR stimulation of MoS2 with human stem cells is investigated using whole-transcriptome sequencing (RNA-seq). Global gene expression profile of stem cells reveals significant influence of MoS2 and NIR stimulation of MoS2 on integrins, cellular migration, and wound healing. The combination of MoS2 and NIR light may provide new approaches to regulate and direct these cellular functions for the purposes of regenerative medicine as well as cancer therapy.

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.

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

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

Enantioselective vinylogous aldol reaction of acylphosphonates with 3-alkylidene oxindoles.

A simple strategy for yielding chiral tertiary α-hydroxy phosphonates that integrates two highly biologically relevant scaffolds namely 3-alkylidene-2-oxindoles and phosphonates has been described. The hydrogen bonding ability of the bifunctional thiourea catalyst allows simultaneous dual activation of a vinylogous oxindole nucleophile and an acylphosphonate electrophile, affording hydroxyphosphonato-3-alkylidene-2-oxindoles as aldol adducts in high yields (up to 92%) with excellent stereocontrol (up to 99% ee).

Widespread changes in transcriptome profile of human mesenchymal stem cells induced by two-dimensional nanosilicates.

Two-dimensional nanomaterials, an ultrathin class of materials such as graphene, nanoclays, transition metal dichalcogenides (TMDs), and transition metal oxides (TMOs), have emerged as a new generation of materials due to their unique properties relative to macroscale counterparts. However, little is known about the transcriptome dynamics following exposure to these nanomaterials. Here, we investigate the interactions of 2D nanosilicates, a layered clay, with human mesenchymal stem cells (hMSCs) at the whole-transcriptome level by high-throughput sequencing (RNA-seq). Analysis of cell-nanosilicate interactions by monitoring changes in transcriptome profile uncovered key biophysical and biochemical cellular pathways triggered by nanosilicates. A widespread alteration of genes was observed due to nanosilicate exposure as more than 4,000 genes were differentially expressed. The change in mRNA expression levels revealed clathrin-mediated endocytosis of nanosilicates. Nanosilicate attachment to the cell membrane and subsequent cellular internalization activated stress-responsive pathways such as mitogen-activated protein kinase (MAPK), which subsequently directed hMSC differentiation toward osteogenic and chondrogenic lineages. This study provides transcriptomic insight on the role of surface-mediated cellular signaling triggered by nanomaterials and enables development of nanomaterials-based therapeutics for regenerative medicine. This approach in understanding nanomaterial-cell interactions illustrates how change in transcriptomic profile can predict downstream effects following nanomaterial treatment.

Regulation of PI4P levels by PI4KIIIα during G-protein-coupled PLC signaling in Drosophila photoreceptors.

The activation of phospholipase C (PLC) is a conserved mechanism of receptor-activated cell signaling at the plasma membrane. PLC hydrolyzes the minor membrane lipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], and continued signaling requires the resynthesis and availability of PI(4,5)P2 at the plasma membrane. PI(4,5)P2 is synthesized by the phosphorylation of phosphatidylinositol 4-phosphate (PI4P). Thus, a continuous supply of PI4P is essential to support ongoing PLC signaling. While the enzyme PI4KA has been identified as performing this function in cultured mammalian cells, its function in the context of an in vivo physiological model has not been established. In this study, we show that, in Drosophila photoreceptors, PI4KIIIα activity is required to support signaling during G-protein-coupled PLC activation. Depletion of PI4KIIIα results in impaired electrical responses to light, and reduced plasma membrane levels of PI4P and PI(4,5)P2 Depletion of the conserved proteins Efr3 and TTC7 [also known as StmA and L(2)k14710, respectively, in flies], which assemble PI4KIIIα at the plasma membrane, also results in an impaired light response and reduced plasma membrane PI4P and PI(4,5)P2 levels. Thus, PI4KIIIα activity at the plasma membrane generates PI4P and supports PI(4,5)P2 levels during receptor activated PLC signaling.

Stereoselective formal [3 + 3] annulation of 3-alkylidene-2-oxindoles with ß,γ-unsaturated α-keto esters.

1,4-Diazabicyclo[2.2.2]octane (DABCO)-catalyzed [3 + 3] cycloaddition reaction of 3-alkylidene-2-oxindole and ß,γ-unsaturated α-keto esters under mild reaction conditions afforded the spirocyclohexene-oxindole with excellent diastereoselectivity. The [3 + 3] annulation is found to proceed through a vinylogous Michael-aldol cascade reaction and it allows rapid access to a diverse set of highly functionalized spirocyclohexene-oxindoles. Also, a bioactivity study of the compounds on mammalian sarcoma cells has reflected cell growth inhibitory/anti-cancer properties.

DYRK1A interacts with histone acetyl transferase p300 and CBP and localizes to enhancers.

DYRK1A, dual-specificity tyrosine phosphorylation-regulated kinase 1A, which is linked to mental retardation and microcephaly, is a member of the CMGC group of kinases. It has both cytoplasmic and nuclear functions, however, molecular mechanisms of how DYRK1A regulates gene expression is not well understood. Here, we identify two histone acetyltransferases, p300 and CBP, as interaction partners of DYRK1A through a proteomics study. We show that overexpression of DYKR1A causes hyperphosphorylation of p300 and CBP. Using genome-wide location (ChIP-sequencing) analysis of DYRK1A, we show that most of the DYRK1A peaks co-localize with p300 and CBP, at enhancers or near the transcription start sites (TSS). Modulation of DYRK1A, by shRNA mediated reduction or transfection mediated overexpression, leads to alteration of expression of downstream located genes. We show that the knockdown of DYRK1A results in a significant loss of H3K27acetylation at these enhancers, suggesting that DYRK1A modulates the activity of p300/CBP at these enhancers. We propose that DYRK1A functions in enhancer regulation by interacting with p300/CBP and modulating their activity. Overall, DYRK1A function in the regulation of enhancer activity provides a new mechanistic understanding of DYRK1A mediated regulation of gene expression, which may help in better understanding of the roles of DYRK1A in human pathologies.

Management of multiloculated hydrocephalus in children with emphasis on role of CT ventriculography.

OBJECTIVE: The authors performed a retrospective review of children diagnosed with multiloculated hydrocephalus (MLH) in our institute. The goal was to analyze the different diagnostic and therapeutic modalities used with special emphasis on CT ventriculography (CTV). METHODS: Male and female patients below the age of 18 years diagnosed with MLH were included. Cases of uniloculated hydrocephalus like entrapped temporal horn or isolated fourth ventricle were excluded. We used iohexol for CTV and gadodiamide for MR ventriculography. Neuroendoscopic procedures performed were endoscopic fenestration, endoscopic third ventriculostomy (ETV), endoscopic septostomy, endoscopic aqueductoplasty, or a combination of the above. The cohort was divided into two groups (endoscopic or shunt) based on initial surgical intervention. RESULTS: A total of 52 patients were included, with 43 boys and 9 girls. The average age of presentation was 7.7 months. The most common predisposing factor for MLH was neonatal meningitis seen in 30 patients. Mean duration of follow-up was 39 months. CTV was used in 26 patients and MR ventriculography in three patients. In one patient, the diagnosis of MLH was ruled out after ventriculography. Patients who underwent ETV only had the best outcome with 71.4% success rate. At the end of follow-up, 14 patients (27%) were shunt independent. CONCLUSIONS: The present study indicates that CTV helps to accurately define the anatomy of the ventricles and determine the site of physiological CSF obstruction. This helps in therapeutic planning and in avoiding misdiagnoses. Further, neuroendoscopy has the potential to lead to shunt independence in some patients.

Ubr3, a Novel Modulator of Hh Signaling Affects the Degradation of Costal-2 and Kif7 through Poly-ubiquitination.

Hedgehog (Hh) signaling regulates multiple aspects of metazoan development and tissue homeostasis, and is constitutively active in numerous cancers. We identified Ubr3, an E3 ubiquitin ligase, as a novel, positive regulator of Hh signaling in Drosophila and vertebrates. Hh signaling regulates the Ubr3-mediated poly-ubiquitination and degradation of Cos2, a central component of Hh signaling. In developing Drosophila eye discs, loss of ubr3 leads to a delayed differentiation of photoreceptors and a reduction in Hh signaling. In zebrafish, loss of Ubr3 causes a decrease in Shh signaling in the developing eyes, somites, and sensory neurons. However, not all tissues that require Hh signaling are affected in zebrafish. Mouse UBR3 poly-ubiquitinates Kif7, the mammalian homologue of Cos2. Finally, loss of UBR3 up-regulates Kif7 protein levels and decreases Hh signaling in cultured cells. In summary, our work identifies Ubr3 as a novel, evolutionarily conserved modulator of Hh signaling that boosts Hh in some tissues.

Traumatic Pediatric Extradural Hematoma: An Institutional Study of 228 Patients in Tertiary Care Center.

BACKGROUND: Extradural hematoma (EDH) is one of the most common causes of mortality and morbidity after traumatic brain injury in pediatric patients. Early surgical intervention in these patients produces excellent results. OBJECTIVE: We reviewed surgical experience at our center, examining and presenting symptomatology and outcome analysis. MATERIALS AND METHODS: A retrospective study of 228 pediatric patients of EDH from July 2007 to August 2017 was performed. Patients were evaluated in terms of demographic profile, clinical features, pupillary size and reaction, computed tomography findings, operative measures, and several other parameters. Neurological status was assessed using motor component (M) of Glasgow Coma Scale score. Best motor response was considered as a criterion to classify severity of traumatic brain injury and for the assessment of outcome. RESULTS: Most of the patients were in the age group of 13-18 years (n = 122, 53.5%). Majority of them were male (n = 182, 79.8%). The commonest mode of injury was fall from height (n = 116, 50.9%) followed by road traffic accident (n = 92, 40.4%). Most common site of hematoma was frontal region (n = 66, 28.9%) followed by parietal region (n = 54, 23.7%). The volume of hematoma was between 30 and 50 mL in majority of the patients (n = 186, 81.6%), and most of the patients had a motor responses of M5 (n = 88, 38.6%) and M6 (n = 108, 47.4%). The association between hematoma site and volume was not significant (χ2 = 5.910, p = 0.749), whereas statistically significant association was noted between volume of hematoma and motor response (χ2 = 93.468, p ≤ 0.001), volume and age (χ2 = 7.380, p ≤ 0.05), and volume to time between trauma and surgery (χ2 = 8.469, p ≤ 0.05). Maximum mortality was in patients of low motor (M1-M3) response and who were operated 24 h after injury. CONCLUSION: Mortality in patients of EDH can be significantly reduced with gratifying results if operated early. Best motor response at presentation, pupillary abnormalities, time between injury to surgery, and location of hematoma have been identified as the important factors determining outcome in patients of EDH.

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.

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.

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.].

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.

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.