RESUMO
The cellular protein homeostasis (proteostasis) network responds effectively to insults. In a functional screen in C. elegans, we recently identified the gene receptor-mediated endocytosis 8 (rme-8; human ortholog: DNAJC13) as a component of the proteostasis network. Accumulation of aggregation-prone proteins, such as amyloid-ß 42 (Aß), α-synuclein, or mutant Cu/Zn-superoxide dismutase (SOD1), were aggravated upon the knockdown of rme-8/DNAJC13 in C. elegans and in human cell lines, respectively. DNAJC13 is involved in endosomal protein trafficking and associated with the retromer and the WASH complex. As both complexes have been linked to autophagy, we investigated the role of DNAJC13 in this degradative pathway. In knockdown and overexpression experiments, DNAJC13 acts as a positive modulator of autophagy. In contrast, the overexpression of the Parkinson's disease-associated mutant DNAJC13(N855S) did not enhance autophagy. Reduced DNAJC13 levels affected ATG9A localization at and its transport from the recycling endosome. As a consequence, ATG9A co-localization at LC3B-positive puncta under steady-state and autophagy-induced conditions is impaired. These data demonstrate a novel function of RME-8/DNAJC13 in cellular homeostasis by modulating ATG9A trafficking and autophagy.
Assuntos
Autofagia , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Chaperonas Moleculares/metabolismo , Proteostase , Animais , Caenorhabditis elegans/citologia , Células HEK293 , Células HeLa , Humanos , Agregados ProteicosRESUMO
One of the numerous functions of glial cells in Drosophila is the ensheathment of neurons to isolate them from the potassium-rich haemolymph, thereby establishing the blood-brain barrier. Peripheral nerves of flies are surrounded by three distinct glial cell types. Although all embryonic peripheral glia (ePG) have been identified on a single-cell level, their contribution to the three glial sheaths is not known. We used the Flybow system to label and identify each individual ePG in the living embryo and followed them into third instar larva. We demonstrate that all ePG persist until the end of larval development and some even to adulthood. We uncover the origin of all three glial sheaths and describe the larval differentiation of each peripheral glial cell in detail. Interestingly, just one ePG (ePG2) exhibits mitotic activity during larval stages, giving rise to up to 30 glial cells along a single peripheral nerve tract forming the outermost perineurial layer. The unique mitotic ability of ePG2 and the layer affiliation of additional cells were confirmed by in vivo ablation experiments and layer-specific block of cell cycle progression. The number of cells generated by this glial progenitor and hence the control of perineurial hyperplasia correlate with the length of the abdominal nerves. By contrast, the wrapping and subperineurial glia layers show enormous hypertrophy in response to larval growth. This characterisation of the embryonic origin and development of each glial sheath will facilitate functional studies, as they can now be addressed distinctively and genetically manipulated in the embryo.
Assuntos
Drosophila/embriologia , Neuroglia/fisiologia , Sistema Nervoso Periférico/embriologia , Animais , Diferenciação Celular/fisiologia , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/imunologia , Proteínas de Homeodomínio/imunologia , Imuno-Histoquímica , Microscopia Confocal , Neuroglia/citologia , Sistema Nervoso Periférico/crescimento & desenvolvimentoRESUMO
Development of the nervous system and establishment of complex neuronal networks require the concerted activity of different signalling events and guidance cues, which include Netrins and their receptors. In Drosophila, two Netrins are expressed during embryogenesis by cells of the ventral midline and serve as attractant or repellent cues for navigating axons. We asked whether glial cells, which are also motile, are guided by similar cues to axons, and analysed the influence of Netrins and their receptors on glial cell migration during embryonic development. We show that in Netrin mutants, two distinct populations of glial cells are affected: longitudinal glia (LG) fail to migrate medially in the early stages of neurogenesis, whereas distinct embryonic peripheral glia (ePG) do not properly migrate laterally into the periphery. We further show that early Netrin-dependent guidance of LG requires expression of the receptor Frazzled (Fra) already in the precursor cell. At these early stages, Netrins are not yet expressed by cells of the ventral midline and we provide evidence for a novel Netrin source within the neurogenic region that includes neuroblasts. Later in development, most ePG transiently express uncoordinated 5 (unc5) during their migratory phase. In unc5 mutants, however, two of these cells in particular exhibit defective migration and stall in, or close to, the central nervous system. Both phenotypes are reversible in cell-specific rescue experiments, indicating that Netrin-mediated signalling via Fra (in LG) or Unc5 (in ePG) is a cell-autonomous effect.
Assuntos
Drosophila/embriologia , Fatores de Crescimento Neural/fisiologia , Neuroglia/fisiologia , Animais , Movimento Celular , Polaridade Celular , Sinais (Psicologia) , Drosophila/genética , Drosophila/fisiologia , Éxons/genética , Imuno-Histoquímica , Mutação , Netrina-1 , Neuroglia/citologia , Fenótipo , Transdução de Sinais , Proteínas Supressoras de Tumor/fisiologiaRESUMO
In the article "Predetermined embryonic glial cells form the distinct glial sheaths of the Drosophila peripheral nervous system" we combined our expertise to identify glial cells of the embryonic peripheral nervous system on a single cell resolution with the possibility to genetically label cells using Flybow. We show that all 12 embryonic peripheral glial cells (ePG) per abdominal hemisegment persist into larval (and even adult) stages and differentially contribute to the three distinct glial layers surrounding peripheral nerves. Repetitive labelings of the same cell further revealed that layer affiliation, morphological expansion, and control of proliferation are predetermined and subject to an intrinsic differentiation program. Interestingly, wrapping and subperineurial glia undergo enormous hypertrophy in response to larval growth and elongation of peripheral nerves, while perineurial glia respond to the same environmental changes with hyperplasia. Increase in cell number from embryo (12 cells per hemisegment) to third instar (up to 50 cells per hemisegment) is the result of proliferation of a single ePG that serves as transient progenitor and only contributes to the outermost perineurial glial layer.
Assuntos
Diferenciação Celular , Drosophila/embriologia , Neuroglia/citologia , Animais , Crescimento Celular , Proliferação de Células , Drosophila/citologia , Mitose , Sistema Nervoso Periférico/citologiaRESUMO
Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. Here we studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, we show that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, we uncovered the origin of each of the peripheral glial cells and linked them to identified central and peripheral neural stem cells.