In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Protein aggregates containing ubiquitinated proteins are commonly present in neurodegenerative disorders and have been considered to cause neuronal degeneration. Here, we report that transient cerebral ischemia caused severe protein aggregation in hippocampal CA1 neurons. By using ethanolic phosphotungstic acid electron microscopy (EM) and ubiquitin immunogold EM, we found that protein aggregates were accumulated in CA1 neurons destined to die 72 hr after 15 min of cerebral ischemia. Protein aggregates appeared as clumps of electron-dense materials that stained heavily for ubiquitin and were associated with various intracellular membranous structures. The protein aggregates appeared at 4 hr and progressively accumulated at 24 and 48 hr of reperfusion in CA1 dying neurons. However, they were rarely observed in dentate gyrus neurons that were resistant to ischemia. At 4 hr of reperfusion, protein aggregates were mainly associated with intracellular vesicles in the soma and dendrites, and the nuclear membrane. By 24 hr of reperfusion, the aggregates were also associated with mitochondria, the Golgi apparatus, and the dendritic plasmalemma. High-resolution confocal microscopy further demonstrated that protein aggregates containing ubiquitin were persistently and progressively accumulated in all CA1 dying neurons but not in neuronal populations that survive in this model. We conclude that proteins are severely aggregated in hippocampal neurons vulnerable to transient brain ischemia. We hypothesize that the accumulation of protein aggregates cause ischemic neuronal death.

Release of cytochrome c (cyt c) into cytoplasm initiates caspase-mediated apoptosis, whereas activation of Akt kinase by phosphorylation at serine-473 prevents apoptosis in several cell systems. To investigate cell death and cell survival pathways, the authors studied release of cyt c, activation of caspase, and changes in Akt phosphorylation in rat brains subjected to 15 minutes of ischemia followed by varying periods of reperfusion. The authors found by electron microscopic study that a portion of mitochondria was swollen and structurally altered, whereas the cell membrane and nuclei were intact in hippocampal CA1 neurons after 36 hours of reperfusion. In some neurons, the pattern of immunostaining for cyt c changed from a punctuate pattern, likely representing mitochondria, to a more diffuse cytoplasmic localization at 36 and 48 hours of reperfusion as examined by laser-scanning confocal microscopic study. Western blot analysis showed that cyt c was increased in the cytosolic fraction in the hippocampus after 36 and 48 hours of reperfusion. Consistently, caspase-3-like activity was increased in these hippocampal samples. As demonstrated by Western blot using phosphospecific Akt antibody, phosphorylation of Akt at serine-473 in the hippocampal region was highly increased during the first 24 hours but not at 48 hours of reperfusion. The authors conclude that transient cerebral ischemia activates both cell death and cell survival pathways after ischemia. The activation of Akt during the first 24 hours conceivably may be one of the factors responsible for the delay in neuronal death after global ischemia.

Transient ischemia leads to changes in synaptic efficacy and results in selective neuronal damage during the postischemic phase, although the mechanisms are not fully understood. The protein composition and ultrastructure of postsynaptic densities (PSDs) were studied by using a rat transient ischemic model. We found that a brief ischemic episode induced a marked accumulation in PSDs of the protein assembly ATPases, N-ethylmaleimide-sensitive fusion protein, and heat-shock cognate protein-70 as well as the BDNF receptor (trkB) and protein kinases, as determined by protein microsequencing. The changes in PSD composition were accompanied by a 2.5-fold increase in the yield of PSD protein relative to controls. Biochemical modification of PSDs correlated well with an increase in PSD thickness observed in vivo by electron microscopy. We conclude that a brief ischemic episode modifies the molecular composition and ultrastructure of synapses by assembly of proteins to the postsynaptic density, which may underlie observed changes in synaptic function and selective neuronal damage.

SUMMARY Chaperones, especially the stress inducible Hsp70, have been studied for their potential to protect the brain from ischemic injury. While they protect from both global and focal ischemia in vivo and cell culture models of ischemia/reperfusion injury in vitro, the mechanism of protection is not well understood. Protein aggregation is part of the etiology of chronic neurodegenerative diseases such as Huntington's and Alzheimer's, and recent data demonstrate protein aggregates in animal models of stroke. We now demonstrate that overexpression of Hsp70 in hippocampal CA1 neurons reduces evidence of protein aggregation under conditions where neuronal survival is increased. We have also demonstrated protection by the cochaperone Hdj-2 in vitro and demonstrated that this is associated with reduced protein aggregation identified by ubiquitin immunostaining. Hdj-2 can prevent protein aggregate formation by itself, but can only facilitate protein folding in conjunction with Hsp70. Pharmacological induction of Hsp70 was found to reduce both apoptotic and necrotic astrocyte death induced by glucose deprivation or oxygen glucose deprivation. Protection from ischemia and ischemia-like injury by chaperones thus involves at least anti-apoptotic,anti-necrotic and anti-protein aggregation mechanisms.

Autophagy is the chief machinery for bulk degradation of superfluous or aberrant cytoplasmic components. This study used the rat moderate fluid percussion injury model to investigate whether the autophagy pathway plays a key role after traumatic brain injury (TBI). Induction of autophagy is manifested by accumulation of autophagosomes (APs), observable under transmission electron microscopy (EM). Two hallmarks of autophagy, i.e., the microtubule-associated protein light chain 3 (LC3)-II and the autophagy-related gene (ATG)12-ATG5 conjugates, were explored by biochemical and confocal microscopic analyses of brain tissues. Under EM, both APs and autolysosomes were markedly accumulated in neurons from 4 h onward after TBI. Western blot analysis showed that ATG12-ATG5 conjugate was markedly redistributed during 5 to 15 days in brain tissues after TBI. LC3-II conjugate was initially unchanged but was drastically upregulated from 24 h onward in the pre-AP-containing fraction after TBI. LC-3 immunostaining was mainly located in living neurons under confocal microscopy. These results clearly show that the autophagy pathway is persistently activated after TBI. Because the autophagy pathway is the chief machinery for bulk elimination of aberrant cell components, we propose that activation of this pathway serves as a protective mechanism for maintaining cellular homeostasis after TBI.

Autophagy is the main degradation pathway responsible for eliminating abnormal protein aggregates and damaged organelles prevalent in neurons after transient cerebral ischemia. This study investigated whether accumulation of protein aggregate-associated organelles in postischemic neurons is a consequence of changes in autophagy. Electron microscopic (EM) analysis indicated that both autophagosomes (AP) and autolysosomes (AL) are significantlly upregulated in hippocampal CA1 and DG neurons after ischemia. The LC3-II conjugate, a marker for APs assessed by Western blotting, was upregulated in postischemic brain tissues. Confocal microscopy showed that LC3 isoforms were located in living postischemic neurons. Treatment with chloriquine (CQ) resulted in accumulation of LC3-II in sham-operated rats, but did not further change the LC3-II levels in postischemic brain tissues. The results indicate that at least part of the accumulation of protein aggregate-associated organelles seen following ischemia is likely to be due to failure of the autophagy pathway. The resulting protein aggregation on subcellular organelle membranes could lead to multiple organelle damage and to delayed neuronal death after transient cerebral ischemia.

Background and Purpose-Protein unfolding and aggregation are dominant early pathogenic events in neurons after brain ischemia. This study used a transient cerebral ischemia model to investigate whether overproduction of unfolded proteins after brain ischemia is a consequence of proteasome dysfunction. Methods-Proteasome peptidase activity and proteasome subcellular redistribution and assembly were studied by peptidase activity assay, Western blot analysis, and size-exclusion chromatography. Results-Proteasome peptidase activity, as determined with the peptide substrate succinyl-LLVY-7-amino-4-methylcoumarin, was moderately decreased, and the 26S proteasome was disassembled during the early period of reperfusion after transient brain ischemia. Furthermore, the proteasome subunits, particularly the 19S components, were deposited into the protein aggregate-containing fraction after an episode of transient cerebral ischemia. Conclusions-These results clearly demonstrate that after an episode of brain ischemia, proteasomes are disassembled and aggregated and thus fail to function normally. Deposition of proteasomes into protein aggregates may also indicate that proteasomes attempt to degrade ubiquitin-conjugated proteins (ubiproteins) overproduced after brain ischemia. However, ubiproteins are too numerous to be degraded and trap some of the proteasomes into their aggregates after brain ischemia. (Stroke. 2007;38:3230-3236.)