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 ubiquitin-proteasome system (UPS) and autophagy-lysosome pathway (ALP) are the two most important mechanisms that normally repair or remove abnormal proteins. Alterations in the function of these systems to degrade misfolded and aggregated proteins are being increasingly recognized as playing a pivotal role in the pathogenesis of many neurodegenerative disorders such as Parkinson's disease. Dysfunction of the UPS has been already strongly implicated in the pathogenesis of this disease and, more recently, growing interest has been shown in identifying the role of ALP in neurodegeneration. Mutations of alpha-synuclein and the increase of intracellular concentrations of non-mutant alpha-synuclein have been associated with Parkinson's disease phenotype. The demonstration that alpha-synuclein is degraded by both proteasome and autophagy indicates a possible linkage between the dysfunction of the UPS or ALP and the occurrence of this disorder. The fact that mutant alpha-synucleins inhibit ALP functioning by tightly binding to the receptor on the lysosomal membrane for autophagy pathway further supports the assumption that impairment of the ALP may be related to the development of Parkinson's disease. In this review, we summarize the recent findings related to this topic and discuss the unique role of the ALP in this neurogenerative disorder and the putative therapeutic potential through ALP enhancement.

Dendritic cells are potent antigen-presenting cells that initiate and amplify immune responses. To determine whether dendritic cells participate in inflammatory reactions in amyotrophic lateral sclerosis (ALS), we examined mRNA expression of dendritic cell surface markers in individual sporadic ALS (sALS), familial ALS (fALS), and nonneurological disease control (NNDC) spinal cord tissues using semiquantitative and real-time reverse transcription polymerase chain reaction (RT-PCR). Immature (DEC205, CD1a) and activated/mature (CD83, CD40) dendritic cell transcripts were significantly elevated in ALS tissues. The presence of immature and activated/mature dendritic cells (CD1a(+) and CD83(+)) was confirmed immunohistochemically in ALS ventral horn and corticospinal tracts. Monocytic/macrophage/microglial transcripts (CD14, CD18, SR-A, CD68) were increased in ALS spinal cord, and activated CD68(+) cells were demonstrated in close proximity to motor neurons. mRNA expressions of the chemokine MCP-1, which attracts monocytes and myeloid dendritic cells, and of the cytokine macrophage-colony stimulating factor (M-CSF) were increased in ALS tissues. The MCP-1 protein was expressed in glia in ALS but not in control tissues and was increased in the CSF of ALS patients. Those patients who progressed most rapidly expressed significantly more dendritic transcripts than patients who progressed more slowly. These results support the involvement of immune/inflammatory responses in amplifying motor neuron degeneration in ALS.

Excessive misfolded proteins and/or dysfunctional mitochondria, which may cause energy deficiency, have been implicated in the etiopathogenesis of Parkinson’s disease (PD). Enhanced clearance of misfolded proteins or injured mitochondria via autophagy has been reported to have neuroprotective roles in PD models. The fact that resveratrol is a known compound with multiple beneficial effects similar to those associated with energy metabolism led us to explore whether neuroprotective effects of resveratrol are related to its role in autophagy regulation. We tested whether modulation of mammalian silent information regulator 2 (SIRT1) and/or metabolic energy sensor AMP-activated protein kinase (AMPK) are involved in autophagy induction by resveratrol, leading to neuronal survival. Our results showed that resveratrol protected against rotenone-induced apoptosis in SH-SY5Y cells and enhanced degradation of α-synucleins in α-synuclein-expressing PC12 cell lines via autophagy induction. We found that suppression of AMPK and/or SIRT1 caused decrease of protein level of LC3-II, indicating that AMPK and/or SIRT1 are required in resveratrol-mediated autophagy induction. Moreover, suppression of AMPK caused inhibition of SIRT1 activity and attenuated protective effects of resveratrol on rotenone-induced apoptosis, further suggesting that AMPK-SIRT1-autophagy pathway plays an important role in the neuroprotection by resveratrol on PD cellular models.

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