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

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Accumulation of unfolded proteins can disturb the functions of the endoplasmic reticulum (ER), leading to ER-stress or unfolded protein response (UPR). Recent data have shown that activation of UPR can be found in postmortem brains of Alzheimer's disease (AD) patients; and biological markers for activation of UPR are abundant in neurons with diffuse phosphorylated tau. Although these observations suggest a linkage between ER-stress and tau pathology, little is known of their relationship. In this study, we found that high levels of phosphorylated PKR-like ER-resident kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) as markers for activation of UPR in the hippocampus of aged P301L mutant tau transgenic mice. The immunoreactivity of p-PERK was found to co-localize with that of phosphorylated tau. We then hypothesized that phosphorylation of tau could induce ER-stress and vice versa in promoting AD-like pathogenesis. By using the protein phosphatase 2A inhibitor okadaic acid (OA) as an inducer for phosphorylation of tau, we found that primary cultures of rat cortical neurons treated with OA triggered UPR as indicated by increased levels of p-PERK and p-eIF2α, splicing of mRNA for xbp-1 and elevated levels of mRNA for GADD153. On the other hand, thapsigargin as an ER-stress inducer stimulated phosphorylation of tau at Thr231, Ser262 and Ser396. Thapsigargin also induced activation of caspase-3 and cleavage of tau. These findings suggested that ER-stress and hyperphosphorylation of tau could be induced by each other to form a vicious cycle to propagate AD-like neurodegeneration.

Abstract. Alzheimer's disease (AD) is an age-related neurodegenerative disease. There are increasing lines of evidence showing that the molecular signaling pathways in aged cells are altered so that cells are susceptible to injury. We and other laboratories have demonstrated the significant involvement of double-stranded RNA-dependent protein kinase (PKR) in ß-amyloid (Aß) peptide neurotoxicity and in AD. Fructus lycii (the fruit of Lycium barbarum) has long been used in oriental medicine as an anti-aging agent. Our previous studies demonstrated that the aqueous extract isolated from L. barbarum exhibited significant protection on cultured neurons against harmful chemical toxins such as Aß and dithiothreitol. We also showed that the polysaccharidecontaining extract (LBP) from L. barbarum exhibited neuroprotective effects in the retina against ocular hypertension in a laser-induced glaucoma animal model. In this study, we aimed to investigate whether LBP can elicit neuroprotection to neurons stressed by Aß peptides. Furthermore, we planned to isolate and identify the neuroprotective agent from LBP using chromatographic methods. Our results showed that pretreatment of LBP effectively protected neurons against Aß-induced apoptosis by reducing the activity of both caspase-3 and -2, but not caspase-8 and -9. A new arabinogalactan-protein (LBP-III) was isolated from LBP and attenuated Aß peptide-activated caspase-3-like activity.LBP-III markedly reduced the phosphorylation of PKR triggered by Aß peptide. Since the phosphorylation state of PKR increased with age, reduction of its phosphorylation triggered by Aß peptide may implicate that LBP-III from Fructus lycii is a potential neuroprotective agent in AD. As herbal medicine has received increasing attention for the treatment of AD, our study will open a window for the development of a neuroprotective agent for anti-aging from Chinese medicine.