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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Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.

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Accumulation of ␣-synuclein (␣-syn) in the brain is a core feature of Parkinson disease (PD) and leads to microglial activation, production of inflammatory cytokines and chemokines, T-cell infiltration, and neurodegeneration. Here, we have used both an in vivo mouse model induced by viral overexpression of ␣-syn as well as in vitro systems to study the role of the MHCII complex in ␣-syn-induced neuroinflammation and neurodegeneration. We find that in vivo, expression of full-length human ␣-syn causes striking induction of MHCII expression by microglia, while knock-out of MHCII prevents ␣-syn-induced microglial activation, antigen presentation, IgG deposition, and the degeneration of dopaminergic neurons. In vitro, treatment of microglia with aggregated ␣-syn leads to activation of antigen processing and presentation of antigen sufficient to drive CD4 T-cell proliferation and to trigger cytokine release. These results indicate a central role for microglial MHCII in the activation of both the innate and adaptive immune responses to ␣-syn in PD and suggest that the MHCII signaling complex may be a target of neuroprotective therapies for the disease.

Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinson's disease. Wild-type α-synuclein and a Parkinson's disease-associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinson's disease.In neurodegenerative diseases, certain populations of adult neurons are gradually lost because of toxic stress. The four myocyte enhancer factor 2 (MEF2) transcription factors, MEF2A to MEF2D, have been shown to play an important role in the survival of several types of neurons, and a genetic polymorphism of the MEF2A gene has been linked to the risk of late onset of Alzheimer's disease (1-3). In cellular models, inhibition of MEF2s contributes to neuronal death. Enhancing MEF2 activity protects neurons from death in vitro and in the substantia nigra pars compacta in a mouse model of Parkinson's disease (PD) (4). Neurotoxic insults cause MEF2 degradation in part by a caspase-dependent mechanism (5), but how MEF2 is regulated under basal conditions without overt toxicity is unknown. Autophagy refers to the degradation of intracellular components by lysosomes. Relative to macro-and microautophagy, chaperonemediated autophagy (CMA) selectively degrades cytosolic proteins (6). This process involves binding of heat shock protein Hsc70 to substrate proteins via a KFERQ-like motif and their subsequent targeting to lysosomes via the lysosomal membrane receptor Lamp2a. Dysregulation of autophagy plays a role in neurodegeneration (7-9). However, the direct mechanism by which CMA modulates neuronal survival or death is unclear.

α-synuclein (α-syn) is a main component of Lewy bodies (LB) that occur in many neurodegenerative diseases, includingParkinson's disease (PD), dementia with LB (DLB) and multi-system atrophy. α-syn mutations or amplifications are responsible for a subset of autosomal dominant familial PD cases, and overexpression causes neurodegeneration and motor disturbances in animals. To investigate mechanisms for α-syn accumulation and toxicity, we studied a mouse model of lysosomal enzyme cathepsin D (CD) deficiency, and found extensive accumulation of endogenous α-syn in neurons without overabundance of α-syn mRNA. In addition to impaired macroautophagy, CD deficiency reduced proteasome activity, suggesting an essential role for lysosomal CD function in regulating multiple proteolytic pathways that are important for α-syn metabolism. Conversely, CD overexpression reduces α-syn aggregation and is neuroprotective against α-syn overexpression-induced cell death in vitro. In a C. elegans model, CD deficiency exacerbates α-syn accumulation while its overexpression is protective against α-syn-induced dopaminergic neurodegeneration. Mutated CD with diminished enzymatic activity or overexpression of cathepsins B (CB) or L (CL) is not protective in the worm model, indicating a unique requirement for enzymatically active CD. Our data identify a conserved CD function in α-syn degradation and identify CD as a novel target for LB disease therapeutics.

Treatment of cells with the macrolide antibiotic bafilomycin A1, an inhibitor of vacuolar (V)-ATPase, or with the lysosomotropic agent chloroquine, has been shown to pharmacologically inhibit autophagy as evidenced by an accumulation of autophagosomes, which in turn causes Bax-dependent apoptosis. However, bafilomycin A1 has also been reported to inhibit chloroquine-induced apoptosis, suggesting a complex interrelationship between these two inhibitors of autophagy. To determine whether the cytoprotective effect of bafilomycin A1 on chloroquine-treated cells was dependent on inhibition of VATPase, we examined the single and combined effects of bafilomycin and chloroquine on cultured cerebellar granule neurons. When added separately, chloroquine or high concentrations of bafilomycin A1 (Ն10 nM) induced a dose-dependent inhibition of autophagy (as measured by an increase in LC3-II, a marker specific for autophagosomes), followed by caspase-3 activation and cell death. When added in combination, bafilomycin A1 potently inhibited chloroquine-induced caspase-3 activity and cell death at concentrations (Յ1 nM) that neither altered vacuolar acidification nor inhibited autophagy. The neuroprotective effects of bafilomycin A1 against chloroquine were substantially greater than those produced by Bax deficiency. Bafilomycin A1-induced neuroprotection seemed to be stimulus-specific, in that staurosporine-induced death was not attenuated by coaddition of bafilomycin A1. Together, these data suggest that in addition to promoting death via inhibition of V-ATPase and autophagy, bafilomycin A1 possesses novel, neuroprotective properties that inhibit Bax-dependent activation of the intrinsic apoptotic pathway resulting from the pharmacological inhibition of autophagy.