The Serine Protease Homolog, Scarface, Is Sensitive to Nutrient Availability and Modulates the Development of the <i>Drosophila</i> Blood-Brain Barrier.

The adaptable transcriptional response to changes in food availability not only ensures animal survival but also lets embryonic development progress. Interestingly, the CNS is preferentially protected from periods of malnutrition, a phenomenon known as "brain sparing." However, the mechanisms that mediate this response remain poorly understood. To get a better understanding of this, we used Drosophila melanogaster as a model, analyzing the transcriptional response of neural stem cells (neuroblasts) and glia of the blood-brain barrier (BBB) from larvae of both sexes during nutrient restriction using targeted DamID. We found differentially expressed genes in both neuroblasts and glia of the BBB, although the effect of nutrient deficiency was primarily observed in the BBB. We characterized the function of a nutritional sensitive gene expressed in the BBB, the serine protease homolog, scarface (scaf). Scaf is expressed in subperineurial glia in the BBB in response to nutrition. Tissue-specific knockdown of scaf increases subperineurial glia endoreplication and proliferation of perineurial glia in the blood-brain barrier. Furthermore, neuroblast proliferation is diminished on scaf knockdown in subperineurial glia. Interestingly, reexpression of Scaf in subperineurial glia is able to enhance neuroblast proliferation and brain growth of animals in starvation. Finally, we show that loss of scaf in the blood-brain barrier increases sensitivity to drugs in adulthood, suggesting a physiological impairment. We propose that Scaf integrates the nutrient status to modulate the balance between neurogenesis and growth of the BBB, preserving the proper equilibrium between the size of the barrier and the brain.SIGNIFICANCE STATEMENT The Drosophila BBB separates the CNS from the open circulatory system. The BBB glia are not only acting as a physical segregation of tissues but participate in the regulation of the metabolism and neurogenesis during development. Here we analyze the transcriptional response of the BBB glia to nutrient deprivation during larval development, a condition in which protective mechanisms are switched on in the brain. Our findings show that the gene scarface reduces growth in the BBB while promoting the proliferation of neural stem, assuring the balanced growth of the larval brain. Thus, Scarface would link animal nutrition with brain development, coordinating neurogenesis with the growth of the BBB

DLGS97/SAP97 is developmentally upregulated and is required for complex adult behaviors and synapse morphology and function

The synaptic membrane-associated guanylate kinase (MAGUK) scaffolding protein family is thought to play key roles in synapse assembly and synaptic plasticity. Evidence supporting these roles in vivo is scarce, as a consequence of gene redundancy in mammals. The genome of Drosophila contains only one MAGUK gene, discs large (dlg), from which two major proteins originate: DLGA [PSD95 (postsynaptic density 95)-like] and DLGS97 [SAP97 (synapse-associated protein)-like]. These differ only by the inclusion in DLGS97 of an L27 domain, important for the formation of supramolecular assemblies. Known dlg mutations affect both forms and are lethal at larval stages attributable to tumoral overgrowth of epithelia. We generated independent null mutations for each, dlgA and dlgS97. These allowed unveiling of a shift in expression during the development of the nervous system: predominant expression of DLGA in the embryo, balanced expression of both during larval stages, and almost exclusive DLGS97 expression in the adult brain. Loss of embryonic DLGS97 does not alter the development of the nervous system. At larval stages, DLGA and DLGS97 fulfill both unique and partially redundant functions in the neuromuscular junction. Contrary to dlg and dlgA mutants, dlgS97 mutants are viable to adulthood, but they exhibit marked alterations in complex behaviors such as phototaxis, circadian activity, and courtship, whereas simpler behaviors like locomotion and odor and light perception are spared. We propose that the increased repertoire of associations of a synaptic scaffold protein given by an additional domain of protein-protein interaction underlies its ability to integrate molecular networks required for complex functions in adult synapses

Predicción de secuencias regulatorias del gen dlg en Drosophila melanogaster y su evaluación in vivo mediante uso de transgénicos

79 p.Un problema importante de la actual era de la genómica es identificar y caracterizar funcionalmente los genes relevantes para una vía o proceso biológico, para esto no sólo es necesario entender su función en un tejido sino también su regulación transcripcional.El gen dlg de Drosophila melanogaster da origen a dos productos proteícos principales: DLGA, similar a la proteína neuronal de mamífero PDS95; y DLGS97, similar a la proteína neuronal y epitelial de mamífero SAP97. DLGA es expresado en epitelio, tejido neuronal y muscular mientras que DLGS97 es expresado en tejido neuronal y muscular, pero no en epitelio. En un orden temporal, DLGA se expresa de manera más marcada en estado de desarrollo temprano en tejido neuronal, a diferencia de DLGS97, el cual, aumenta sus niveles de expresión en este tejido en estado de mosca adulta. Estos dos transcritos se originan por inicios de transcripción alternativos lo que sugiere una regulación compleja donde múltiples Factores de Transcripción (FT) podrían estar participando. El objetivo de esta investigación es la identificación de la arquitectura de los promotores y los módulos regulatorios del gen dlg. Se pondrá énfasis en la identificación de los módulos de regulación que podrían explicar la expresión del gen neuronal. Un módulo de regulación es una región de ADN en la cual se unen múltiples FT y su influencia combinada produce un patrón de expresión específico. La hipótesis es que estos módulos se encuentran en regiones cercanas a los inicios de transcripción. El objetivo del trabajo es identificar los módulos (conjunto de motivos) que caracteriza cada uno de los patrones de expresión. Para ésta identificación se realizó un enfoque bioinformático de varios pasos que comienza con la comparación genómica de la secuencia de dlg de doce ortólogos disponibles de la familia Drosophilidae para seleccionar los motivos conservados. La selección de motivos conservados se comparó con bases de datos de Sitios de Unión (SU) de FT conocidos. Después de caracterizar los supuestos módulos de regulación del gen dlg se procedió a la búsqueda en todo el genoma para identificar genes que comparten una regulación similar, en particular la búsqueda de genes de expresión neuronal. Finalmente, con las predicciones in-silico se crearon vectores experimentales con los putativos módulos río arriba y la secuencia nuclear GFP con la finalidad de probar in vivo la capacidad del módulo para conducir la expresión en un tejido específico. Se determinaron 4 módulos que se probaron de forma independiente experimentalmente. Dos módulos estarían regulando DLGS97 y los otros dos a DLGA. Los módulos 1 y 2 en embriones transgénicos de Drosophila melanogaster estarían regulando la expresión de DLGA, y los módulos 3 y 4 la expresión de DLGS97. En larvas transgénicas de Drosophila melanogaster los módulos 1 y 4 estarían regulando la expresión de DLGA. En adultos transgénicos los módulos 1, 2 y 4 lo harían para la isoforma DLGS97. Las conclusiones se obtuvieron después de disectar cada uno de los estados de desarrollo de Drosophila melanogaster y ser observados en microscopios de fluorescencia./ABSTRACT: An important subject in the current genomics era is to identify and functionally characterize relevant genes for a specific pathway or biological process, for this is not necessary to understand their function in a tissue but also its transcriptional regulation.The dlg gene of Drosophila melanogaster gives rise to two major protein products: DLGA, similar to neuronal protein of mammal PDS95, and DLGS97, similar to neuronal and epithelial protein of mammal SAP97. DLGA is expressed in epithelial, neuronal and muscle tissues while DLGS97 is expressed in neuronal and muscle tissue but not epithelial. In a temporary order, DLGA is expressed more strongly in early development in neural tissue, unlike DLGS97, which increases their levels of expression in this tissue in the adult fly. These two transcripts originate from alternative transcription starts suggesting a complex regulation where multiple transcription factors could be involved. The aim of this research is to identify the architecture of the promoters and gene regulatory modules of the dlg gene. We will emphasize in the identification of regulatory modules that might explain the neuronal expression of the gene. A regulatory module is a region of DNA that binds multiple transcription factors, and in which their combined influence produces a specific expression pattern. The hypothesis is that these modules are in regions close to the transcription start. The aim of this work is to identify the modules (set of motifs), that characterize each of the patterns of expression. For this identification we performed a bioinformatic approach in multiple steps that starts with a comparison of the dlg genomic sequence of the twelve orthologs from the available genomes of the Drosophilidae family to select conserved motifs. The selected conserved motifs were compared to databases of known transcription factors binding sites. Having characterized the putative gene regulatory modules of dlg, we proceeded to search in the whole genome to identify genes that share a similar regulation, in particular we search for genes of neuronal expression. Finally using in-silico predictions, a vector with the putative modules upstream and the nuclear GFP sequence will be created in order to test in vivo the module capacity to drive expression in a specific tissue.We found 4 modules independently tested experimentally. Two modules that would regulate DLGS97 and the others two DLGA. Modules 1 and 2 in transgenic embryos of Drosophila melanogaster would regulate the expression of DLGA and modules 3 and 4 the DLGS97 expression. In transgenic Drosophila melanogaster larvae, modules 1 and 4 would regulate expression of DLGA. In adult transgenic modules 1, 2 and 4, would regulate the isoform DLGS97. The conclusions were obtained after dissecting each of the stages of development of Drosophila melanogaster and by observation in fluorescence microscop

The Fly Blood-Brain Barrier Fights Against Nutritional Stress

In the wild, animals face different challenges including multiple events of food scarcity. How they overcome these conditions is essential for survival. Thus, adaptation mechanisms evolved to allow the development and survival of an organism during nutrient restriction periods. Given the high energy demand of the nervous system, the molecular mechanisms of adaptation to malnutrition are of great relevance to fuel the brain. The blood-brain barrier (BBB) is the interface between the central nervous system (CNS) and the circulatory system. The BBB mediates the transport of macromolecules in and out of the CNS, and therefore, it can buffer changes in nutrient availability. In this review, we collect the current evidence using the fruit fly, Drosophila melanogaster , as a model of the role of the BBB in the adaptation to starvation. We discuss the role of the Drosophila BBB during nutrient deprivation as a potential sensor for circulating nutrients, and transient nutrient storage as a regulator of the CNS neurogenic niche

How doth the little busy bee: unexpected metabolism

Brain energy metabolism powers information processing and behavior, much as electricity powers a computer. However, a recent study in insects suggests that this relationship is more interesting, causally linking aggressive behavior to energetics. These findings may also shed new light on aerobic glycolysis, a long-standing riddle of human brain physiology

Functionally heterogenous ryanodine receptors in avian cerebellum

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One popul

p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development.

Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, we hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction.Here, we studied the function of p53 during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. We showed that p53 loss of function reduced animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, we showed that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals.Using genetic approaches, we demonstrate that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals

p53 is required for brain growth but is dispensable for resistance to nutrient restriction during Drosophila larval development

Background Animal growth is influenced by the genetic background and the environmental circumstances. How genes promote growth and coordinate adaptation to nutrient availability is still an open question. p53 is a transcription factor that commands the cellular response to different types of stresses. In adult Drosophila melanogaster, p53 regulates the metabolic adaptation to nutrient restriction that supports fly viability. Furthermore, the larval brain is protected from nutrient restriction in a phenomenon called 'brain sparing'. Therefore, we hypothesised that p53 may regulate brain growth and show a protective role over brain development under nutrient restriction. Results Here, we studied the function of p53 during brain growth in normal conditions and in animals subjected to developmental nutrient restriction. We showed that p53 loss of function reduced animal growth and larval brain size. Endogenous p53 was expressed in larval neural stem cells, but its levels and activity were not affected by nutritional stress. Interestingly, p53 knockdown only in neural stem cells was sufficient to decrease larval brain growth. Finally, we showed that in p53 mutant larvae under nutrient restriction, the energy storage levels were not altered, and these larvae generated adults with brains of similar size than wild-type animals. Conclusions Using genetic approaches, we demonstrate that p53 is required for proper growth of the larval brain. This developmental role of p53 does not have an impact on animal resistance to nutritional stress since brain growth in p53 mutants under nutrient restriction is similar to control animals.Fondo Nacional de Desarrollo Cientifico y Tecnologico FONDECYT 3160412 1140522 1171800 Anillo de Investigacion en Ciencia y Tecnologia DRiDANS ACT1401 Institute Milenio de Neurociencia Biomedica (BNI) ICM P09015-F Fondo de Financiamiento de Centros de Investigacion en Areas Prioritarias (Fondap) 1509000

Phospholipase C activity in membranes and a soluble fraction isolated from frog skeletal muscle

Highly purified triads and transverse tubules, as well as a soluble fraction isolated from frog skeletal muscle, hydrolyze exogenous phosphatidylinositol 4,5-bisphosphate forming inositol 1,4,5-trisphosphate with maximal rates in the range 0.5-1 nmol/mg per min at pCa 3. Sarcoplasmic reticulum membranes present a minor activity. The hydrolysis rates in triads were 0.072 ± 0.015 nmol/mg per min at pCa 7, increasing to 0.263 ± 0.026 nmol/mg per min at pCa 5 with 1.0 mM Mg and 0.1 mM substrate. The phospholipase C activity of isolated transverse tubules at pCa 3 was 0.570 ± 0.032 nmol/mg per min. Since triads contain 10% transverse tubules, and correcting for the small contribution of sarcoplasmic reticulum, the calculated phospholipase C activity of transverse tubules at pCa 3 is about 10-times higher than the observed values, suggesting loss of activity during isolation. The activation by calcium was also observed in a soluble fraction and was neither replaced nor inhibited by magnesiu