Establishment of a Novel Fluorescence-Based Method to Evaluate Chaperone-Mediated Autophagy in a Single Neuron

Background: Chaperone-mediated autophagy (CMA) is a selective autophagy-lysosome protein degradation pathway. The role of CMA in normal neuronal functions and in neural disease pathogenesis remains unclear, in part because there is no available method to monitor CMA activity at the single-cell level. Methodology/Principal Findings: We sought to establish a single-cell monitoring method by visualizing translocation of CMA substrates from the cytosol to lysosomes using the HaloTag (HT) system. GAPDH, a CMA substrate, was fused to HT (GAPDH-HT); this protein accumulated in the lysosomes of HeLa cells and cultured cerebellar Purkinje cells (PCs) after labeling with fluorescent dye-conjugated HT ligand. Lysosomal accumulation was enhanced by treatments that activate CMA and prevented by siRNA-mediated knockdown of LAMP2A, a lysosomal receptor for CMA, and by treatments that inactivate CMA. These results suggest that lysosomal accumulation of GAPDH-HT reflects CMA activity. Using this method, we revealed that mutant cPKC, which causes spinocerebellar ataxia type 14, decreased CMA activity in cultured PCs. Conclusion/Significance: In the present study, we established a novel fluorescent-based method to evaluate CMA activity in a single neuron. This novel method should be useful and valuable for evaluating the role of CMA in various neurona

Excess α-synuclein worsens disease in mice lacking ubiquitin carboxy-terminal hydrolase L1

Mutations in α-synuclein (αSN) and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) have been linked to familial Parkinson's disease (PD). Physical and functional interactions between these two proteins have been described. Whether they act additively in vivo to influence disease has remained controversial. αSN is a presynaptic protein and the major constituent of Lewy inclusions, histopathological hallmarks of PD. UCH-L1 regulates ubiquitin stability in the nervous system and its loss results in neurodegeneration in peripheral and central neurons. Here, we used genetics to show that UCH-L1-deficiency together with excess αSN worsen disease. Double mutant mice show earlier-onset motor deficits, a shorter lifespan and forebrain astrogliosis but the additive disease-worsening effects of UCH-L1-deficiency and excess αSN are not accompanied by microgliosis, ubiquitin pathology or changes in pathological αSN protein levels and species

Cytosolic Superoxide Dismutase (SOD1) Is Critical for Tolerating the Oxidative Stress of Zinc Deficiency in Yeast

Zinc deficiency causes oxidative stress in many organisms including the yeast Saccharomyces cerevisiae. Previous studies of this yeast indicated that the Tsa1 peroxiredoxin is required for optimal growth in low zinc because of its role in degrading H2O2. In this report, we assessed the importance of other antioxidant genes to zinc-limited growth. Our results indicated that the cytosolic superoxide dismutase Sod1 is also critical for growth under zinc-limiting conditions. We also found that Ccs1, the copper-delivering chaperone required for Sod1 activity is essential for optimal zinc-limited growth. To our knowledge, this is the first demonstration of the important roles these proteins play under this condition. It has been proposed previously that a loss of Sod1 activity due to inefficient metallation is one source of reactive oxygen species (ROS) under zinc-limiting conditions. Consistent with this hypothesis, we found that both the level and activity of Sod1 is diminished in zinc-deficient cells. However, under conditions in which Sod1 was overexpressed in zinc-limited cells and activity was restored, we observed no decrease in ROS levels. Thus, these data indicate that while Sod1 activity is critical for low zinc growth, diminished Sod1 activity is not a major source of the elevated ROS observed under these conditions

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

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. For example, 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 versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost 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 fi eld 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. 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

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field