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Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons.

Zhang X, Wei M, Fan J, Yan W, Zha X, Song H, Wan R, Yin Y, Wang W
Autophagy 2021
Open on PubMed

1. Autophagy. 2021 Jun;17(6):1519-1542. doi: 10.1080/15548627.2020.1840796. Epub 2020 Nov 12. Ischemia-induced upregulation of autophagy preludes dysfunctional lysosomal storage and associated synaptic impairments in neurons. Zhang X(1), Wei M(1), Fan J(1), Yan W(1), Zha X(1), Song H(1), Wan R(1), Yin Y(1), Wang W(1). Author information: (1)Department of Physiology and Pathophysiology, Department of Neurobiology, Key Laboratory for Neurodegenerative Disorders of the Ministry of Education, School of Basic Medical Sciences, Capital Medical University, Beijing, PR China. Macroautophagy/autophagy is vital for neuronal homeostasis and functions. Accumulating evidence suggest that autophagy is impaired during cerebral ischemia, contributing to neuronal dysfunction and neurodegeneration. However, the outcomes after transient modification in autophagy machinery are not fully understood. This study investigated the effects of ischemic stress on autophagy and synaptic structures using a rat model of oxygen-glucose deprivation (OGD) in hippocampal neurons and a mouse model of middle cerebral artery occlusion (MCAO). Upon acute ischemia, an initial autophagy modification occurred in an upregulation manner. Following, the number of lysosomes increased, as well as lysosomal volume, indicating dysfunctional lysosomal storage. These changes were prevented by inhibiting autophagy via 3-methyladenine (3-MA) treatment or ATG7 (autophagy related 7) knockdown, or were mimicked by rapamycin (RAPA), a known activator of autophagy. This suggests that dysfunctional lysosomal storage is associated with the early burst of autophagy. Dysfunctional lysosomal storage contributed to autophagy dysfunction because the basal level of MTOR-dependent lysosomal biogenesis in the reperfusion was not sufficient to clear undegraded cargoes after transient autophagy upregulation. Further investigation revealed that impairment of synaptic ultra-structures, accompanied by dysfunctional lysosomal storage, may result from a failure in dynamic turnover of synaptic proteins. This indicates a vital role of autophagy-lysosomal machinery in the maintenance of synaptic structures. This study supports previous evidence that dysfunctional lysosomal storage may occur following the upregulation of autophagy in neurons. Appropriate autophagosome-lysosomal functioning is vital for maintenance of neuronal synaptic function and impacts more than the few known synaptic proteins.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin beta; AD: Alzheimer disease; ALR: autophagic lysosome reformation; ATG7: autophagy related 7; CTSB: cathepsin B; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DEGs: differentially expressed genes; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GO: Gene Ontology; HBSS: Hanks' balanced salt solution; HPCA: hippocalcin; i.c.v: intracerebroventricular; KEGG: kyoto encyclopedia of genes and genomes; LAMP1: lysosomal-associated membrane protein 1; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; LSDs: lysosomal storage disorders; MAP2: microtubule-associated protein 2; MCAO: middle cerebral artery occlusion; mCTSB: mature CTSB; mCTSD: mature CTSD; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; OGD/R: oxygen-glucose deprivation/reoxygenation; PBS: phosphate-buffered saline; PRKAA/AMPKα: protein kinase AMP-activated catalytic subunit alpha; proCTSD: pro-cathepsin D; RAPA: rapamycin; RNA-seq: RNA sequencing; RPS6KB/p70S6K: ribosomal protein S6 kinase; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIM: Structured Illumination Microscopy; SNAP25: synaptosomal-associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYT1: synaptotagmin I; TBST: tris-buffered saline Tween-20; TEM: transmission electron microscopy; TFEB: transcription factor EB; tMCAO: transient middle cerebral artery occlusion; TTC: 2,3,5-triphenyltetrazolium chloride; TUBB3: tubulin, beta 3 class III. DOI: 10.1080/15548627.2020.1840796 PMCID: PMC8205014 PMID: 33111641 [Indexed for MEDLINE] Conflict of interest statement: The authors declare no competing financial interests.

11 Figures Extracted
Figure 1.
Figure 1. PMC
Autophagic dysfunction is transiently induced by OGD insult, and a prolonged increase in LAMP1 expression occurs during the subsequent reperfusion. (A...
Figure 2.
Figure 2. PMC
During reperfusion, lysosomal dysfunction may result from reduced CTSD activity. (A) LAMP1-positive puncta in the indicated groups were observed with ...
Figure 3.
Figure 3. PMC
The number and volume of lysosomes increased in cultured hippocampal neurons after 1 h OGD/R 24 h exposure. (A) Representative SIM images of hippocamp...
Figure 4.
Figure 4. PMC
Suppression of OGD-induced autophagy upregulation by 3-MA pretreatment or ATG7 genetic knockdown prevents lysosomal accumulation during the subsequent...
Figure 5.
Figure 5. PMC
RAPA-induced autophagy upregulation is followed by lysosomal accumulation in the subsequent culture without RAPA. (A-C) Representative western blots a...
Figure 6.
Figure 6. PMC
Hippocampal neuronal processes show dysfunctional autophagy after OGD and subsequent reperfusion, which can be disrupted by 3-MA pretreatment and mimi...
Figure 7.
Figure 7. PMC
Synaptic structures are altered after 1 h OGD/24 h reperfusion; these changes are prevented by 3-MA treatment and partially mimicked by RAPA treatment...
Figure 8.
Figure 8. PMC
The turnover of synaptic proteins is impaired after 1 h OGD/24 h reperfusion; these changes are partially mimicked by RAPA treatment. (A) Heat map of ...
Figure 9.
Figure 9. PMC
Lysosome dysfunction is transiently induced by 1 h MCAO insult during subsequent reperfusion in vivo . (A) Representative images of TTC-stained cereb...
Figure 10.
Figure 10. PMC
3-MA pretreatment via the intracerebroventricular (i.c.v) injection alleviated ischemic injuries induced by 1 h MCAO insults during subsequent reperfu...
Figure 11.
Figure 11. PMC
Unmatched activation levels of MTOR may contribute to lysosomal accumulation during subsequent reperfusion in in vitro and in vivo experiments. (A...
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