Dataset Information

Measurement of autophagy flux in the nervous system in vivo.

ABSTRACT: Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulate autophagy have been shown to provide protection against neurodegeneration. Here we describe a novel and simple strategy to express an autophagy flux reporter in the nervous system of adult animals by the intraventricular delivery of adeno-associated viruses (AAV) into newborn mice. Using this approach we efficiently expressed a monomeric tandem mCherry-GFP-LC3 construct in neurons of the peripheral and central nervous system, allowing the measurement of autophagy activity in pharmacological and disease settings.

SUBMITTER: Castillo K

PROVIDER: S-EPMC3847309 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Measurement of autophagy flux in the nervous system in vivo.

Castillo K K Valenzuela V V Matus S S Nassif M M Oñate M M Fuentealba Y Y Encina G G Irrazabal T T Parsons G G Court F A FA Schneider B L BL Armentano D D Hetz C C

Cell death & disease 20131114

Accurate methods to measure autophagic activity in vivo in neurons are not available, and most of the studies are based on correlative and static measurements of autophagy markers, leading to conflicting interpretations. Autophagy is an essential homeostatic process involved in the degradation of diverse cellular components including organelles and protein aggregates. Autophagy impairment is emerging as a relevant factor driving neurodegeneration in many diseases. Moreover, strategies to modulat ...[more]

PMID: 24232093

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Project description:Much of our understanding of the intracellular regulation of macroautophagy/autophagy comes from in vitro studies. However, there remains a paucity of knowledge about how this process is regulated within different tissues during development, aging and disease in vivo. Because upregulation of autophagy is considered a promising therapeutic strategy for the treatment of diverse disorders, it is vital that we understand how this pathway functions in different tissues and this is best done by in vivo analysis. Similarly, to understand the role of autophagy in the pathogenesis of disease, it is important to study this process in the whole animal to investigate how tissue-specific changes in flux and cell-autonomous versus non-cell-autonomous effects alter disease progression. To this end, we have developed an inducible expression system to up- or downregulate autophagy in vivo, in zebrafish. We have used a modified version of the Gal4-UAS expression system to allow inducible expression of autophagy up- or downregulating transgenes by addition of tamoxifen. Using this inducible expression system, we have tested which transgenes robustly up- or downregulate autophagy and have validated these tools using Lc3-II blots and puncta analysis and disease rescue in a zebrafish model of neurodegeneration. These tools allow the temporal control of autophagy via the administration of tamoxifen and spatial control via tissue or cell-specific ERT2-Gal4 driver lines and will enable the investigation of how cell- or tissue-specific changes in autophagic flux affect processes such as aging, inflammation and neurodegeneration in vivo.Abbreviations: ANOVA: analysis of variance; Atg: autophagy related; Bcl2l11/Bim: BCL2 like 11; d.p.f.: days post-fertilization; Cryaa: crystallin, alpha a: DMSO: dimethyl sulfoxide; Elavl3: ELAV like neuron-specific RNA binding protein 3; ER: estrogen receptor; ERT2: modified ligand-binding domain of human ESR1/estrogen receptor α; Gal4: galactose-responsive transcription factor 4; GFP: green fluorescent protein; h.p.f.: hours post-fertilization; HSP: heat-shock protein; Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; RFP: red fluorescent protein; SD: standard deviation; SEM: standard error of the mean; UAS: upstream activating sequence; Ubb: ubiquitin b.

Dataset Information

Measurement of autophagy flux in the nervous system in vivo.

Publications

Measurement of autophagy flux in the nervous system in vivo.

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