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

ABSTRACT

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.