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
Mitochondria continuously undergo two opposing processes, fission and fusion. The disruption of this dynamic equilibrium may herald cell injury or death and may contribute to developmental and neurodegenerative disorders. Nitric oxide functions as a signaling molecule, but in excess it mediates neuronal injury, in part via mitochondrial fission or fragmentation. However, the underlying mechanism for nitric oxide-induced pathological fission remains unclear. We found that nitric oxide produced in response to β-amyloid protein, thought to be a key mediator of Alzheimer's disease, triggered mitochondrial fission, synaptic loss, and neuronal damage, in part via S-nitrosylation of dynamin-related protein 1 (forming SNO-Drp1). Preventing nitrosylation of Drp1 by cysteine mutation abrogated these neurotoxic events. SNO-Drp1 is increased in brains of human Alzheimer's disease patients and may thus contribute to the pathogenesis of neurodegeneration.Disrupting the balance between mitochondrial fission and fusion can lead to excessive mitochondrial fragmentation. Fragmentation triggered by dysfunction of the fission-inducing protein Drp1 (dynamin-related protein 1), for example, contributes to synaptic damage and subsequent neuronal loss because of nitrosative/oxidative stress and impaired bioenergetics (1-6). Excessive fission results in abnormally small mitochondria with fragmented cristae (2), as observed in electron microscopy studies of neurons in human Alzheimer's disease (AD) (7). Drp1 homologs are S-nitrosylated, which regulates their activity (8,9). Furthermore, β-amyloid protein (Aβ) oligomers induce excessive mitochondrial fission and neuronal damage in a nitric oxide (NO)-mediated fashion (2,10). We sought to determine whether Drp1 is Snitrosylated and thereby activated in AD. §To whom correspondence should be addressed. Cerebrocortical neurons transfected with the mitochondrial marker mito-DsRed2 were exposed to the NO donor S-nitrosocysteine (SNOC) (11) and morphological changes in mitochondria were monitored by 3D-deconvolution fluorescence microscopy (2). Mitochondria normally displayed an elongated filamentous morphology, but addition of SNOC induced fragmented, smaller mitochondria in a dose-dependent manner, due to fission (Fig. 1, A and B) (2,11). Using a biotin-switch assay (12), we found that SNOC induced S-nitrosylation of Drp1 (forming SNO-Drp1) in neurons before inducing fission (Fig. 1C).To investigate whether endogenously generated NO can induce SNO-Drp1, we used human embryonic kidney (HEK) 293 cells stably expressing neuronal NO synthase (nNOS). These cells were subjected to biotin-switch assay after incubation with the calcium ionophore A23187 to activate nNOS. Endogenous Drp1 was S-nitrosylated by endogenous NO; this reaction was blocked by the NOS inhibitor N-nitro-L-arginine (NNA; Fig. 2, A and B). SNO-Drp1 was not detected in controls performed without ascorbate to remove NO, thus preventing replacement of NO by biotin (which is detected in this assay), or without biotin-HPDP (N-[...
Mitochondria are highly dynamic organelles that continuously undergo two opposite processes, fission and fusion. Mitochondrial dynamics influence not only mitochondrial morphology, but also mitochondrial biogenesis, mitochondrial distribution within the cell, cell bioenergetics, and cell injury or death. Drp1 mediates mitochondrial fission, whereas Mfn1/2 and Opa1 control mitochondrial fusion. Neurons require large amounts of energy to carry out their highly specialized functions. Thus, mitochondrial dysfunction is a prominent feature in a variety of neurodegenerative diseases. Mutations of Mfn2 and Opa1 lead to neuropathies such as Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. Moreover, both Aβ peptide and mutant huntingtin protein induce mitochondrial fragmentation and neuronal cell death. In addition, mutants of Parkinson's disease-related genes also show abnormal mitochondrial morphology. This review highlights our current understanding of abnormal mitochondrial dynamics relevant to neuronal synaptic loss and cell death in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease and Huntington's disease.
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