Transgenic expression of a ratiometric autophagy probe specifically in neurons enables the interrogation of brain autophagy in vivo

Autophagy. 2019 Mar;15(3):543-557. doi: 10.1080/15548627.2018.1528812. Epub 2018 Oct 26.

Abstract

Autophagy-lysosome pathway (ALP) disruption is considered pathogenic in multiple neurodegenerative diseases; however, current methods are inadequate to investigate macroautophagy/autophagy flux in brain in vivo and its therapeutic modulation. Here, we describe a novel autophagy reporter mouse (TRGL6) stably expressing a dual-fluorescence-tagged LC3 (tfLC3, mRFP-eGFP-LC3) by transgenesis selectively in neurons. The tfLC3 probe distributes widely in the central nervous system, including spinal cord. Expression levels were similar to endogenous LC3 and induced no detectable ALP changes. This ratiometric reporter registers differential pH-dependent changes in color as autophagosomes form, fuse with lysosomes, acidify, and degrade substrates within autolysosomes. We confirmed predicted changes in neuronal autophagy flux following specific experimental ALP perturbations. Furthermore, using a third fluorescence label in TRGL6 brains to identify lysosomes by immunocytochemistry, we validated a novel procedure to detect defective autolysosomal acidification in vivo. Thus, TRGL6 mice represent a unique tool to investigate in vivo ALP dynamics in specific neuron populations in relation to neurological diseases, aging, and disease modifying agents. Abbreviations: ACTB: actin, beta; AD: Alzheimer disease; AL: autolysosomes; ALP: autophagy-lysosome pathway; AP: autophagosome; APP: amyloid beta (Abeta) precursor protein; ATG5: autophagy related 5; ATG7: autophagy related 7; AV: autophagic vacuoles; CNS: central nervous system; CTSD: cathepsin D; CQ: chloroquine; DMEM: Dulbecco's modified Eagle's medium; GFP: green fluorescent protein; GABARAP: gamma-aminobutyric acid receptor associated protein; GABARAPL2/GATE16: gamma-aminobutyric acid (GABA) receptor-associated protein-like 2; ICC: immunocytochemistry; ICV: intra-cerebroventricular; LAMP2: lysosomal-associated membrane protein 2; Leup: leupeptin; LY: lysosomes; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; RFP: red fluorescent protein; RPS6KB1: ribosomal protein S6 kinase, polypeptide 1; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SQSTM1: sequestosome 1; tfLC3: mRFP-eGFP-LC3; TRGL6: Thy1 mRFP eGFP LC3-line 6; PCR: polymerase chain reaction; PD: Parkinson disease.

Keywords: Acidification; autophagosome; cathepsin; lysosome; microtubule associated protein 1 light chain 3 (LC3); neurodegeneration; pH.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagosomes / drug effects
  • Autophagosomes / metabolism
  • Autophagy* / drug effects
  • Autophagy* / genetics
  • Brain / cytology
  • Brain / metabolism*
  • Brain Chemistry
  • Cells, Cultured
  • Chloroquine / pharmacology
  • Fluorescent Dyes
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Lysosomes / chemistry*
  • Lysosomes / drug effects
  • Lysosomes / metabolism
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Microtubule-Associated Proteins / genetics*
  • Microtubule-Associated Proteins / metabolism
  • Morpholines / pharmacology
  • Neurons / chemistry
  • Neurons / cytology
  • Neurons / drug effects
  • Neurons / metabolism*
  • Red Fluorescent Protein

Substances

  • Fluorescent Dyes
  • Luminescent Proteins
  • Map1lc3b protein, mouse
  • Microtubule-Associated Proteins
  • Morpholines
  • Green Fluorescent Proteins
  • Chloroquine
  • (5-(2,4-bis((3S)-3-methylmorpholin-4-yl)pyrido(2,3-d)pyrimidin-7-yl)-2-methoxyphenyl)methanol