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

The data indicate that in the retina RBPMS is selectively expressed in RGCs and therefore could serve as a marker for RGC quantification in normal retinas and for estimation of RGC loss in ocular neuropathies.

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Rbpms can reliably be used as an RGC marker for quantitative evaluation in rat models of RGC degeneration, regardless of the nature and the location of the primary site of the injury and the extent of neurodegeneration.

Autophagy has been shown to be activated in neuronal cells in response to injury and suggested to have a cell-protective role in neurodegenerative diseases. In this study, we investigated the activation of autophagy in retinal ganglion cells (RGCs) following optic nerve transection (ONT) and evaluated its effect on RGC survival. Expression of several autophagy-related genes, including Atg5, Atg7, and Atg12, and autophagy markers microtubule-associated protein 1 light chain 3-II (LC3-II) and beclin-1 were analyzed at the transcriptional or protein level 1, 3, and 7 days after ONT. Transcription of the Atg5, Atg7, and Atg12 genes was upregulated 1.5-to 1.8-fold in the retina 3 days after ONT compared with that in the controls. Expression of Atg12 mRNA was increased 1.6-fold 1 day after ONT. Seven days after ONT, expression of Atg5, Atg7, and Atg12 mRNA was comparable to that in the untreated retinas. Western blot analysis of proteins isolated from RGCs showed 1.6-, 2.7-, and 1.7-fold increases in LC3-II level 1, 3, and 7 days after ONT, respectively, compared with those in the controls. Expression of beclin-1 was 1.7-fold higher 1 day after RGCs were axotomized, but 3 and 7 days after ONT it was comparable to that of the control. Inhibition of autophagy with bafilomycin A1, 3-methyladenine, and Wortmannin in RGC-5 cells under serum-deprived conditions decreased cell viability by approximately 40%. These results suggest possible activation of autophagy in RGCs after optic nerve transection and demonstrate its protective role in RGC-5 cells maintained under conditions of serum deprivation. V V C 2008 Wiley-Liss, Inc.

Increases of approximately 95% and 75% in RGC survival mediated by alphaA and alphaB overexpression, respectively, were observed 14 days after ONT. At day 7, the RGC protective effect of alphaA and alphaB overexpression was approximately 40%.

We investigated the neuroprotective effect of thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2) which play critical roles in the regulation of oxidative stress on retinal ganglion cells (RGCs) in a rat glaucoma model. Expression of Trx1 and Trx2 and Trx-interacting protein (Txnip) was observed in the RGC layer (GCL), nerve fiber layer and inner nuclear layer. Txnip-, Trx1-and Trx2-expressing cells in the GCL were primarily colocalized with RGCs. The increased Txnip protein level was observed 2 and 5 weeks after glaucoma induction. Trx1 level decreased 2 weeks after glaucoma induction and more prominently after 5 weeks. No change in Trx2 levels was detected. The effects of Trx1 and Trx2 overexpression on RGC survival were evaluated 5 weeks after glaucoma induction. In nontransfected and EGFP-transfected (used as a negative control) retinas, RGC loss was approximately 27% compared with control. The loss of RGCs in Trx1-and Trx2-transfected retinas was approximately 15 and 17%, respectively. Thus, Trx1 and Trx2 preserved 45 and 37% of cells, respectively that were destined to die in glaucomatous retinas. The results of this study provide evidence for the involvement of oxidative stress in RGC degeneration in experimental glaucoma and point to potential strategies to reduce its impact.