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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There are several interrelated mechanisms involving iron, dopamine, and neuromelanin in neurons. Neuromelanin accumulates during aging and is the catecholamine-derived pigment of the dopamine neurons of the substantia nigra and norepinephrine neurons of the locus coeruleus, the two neuronal populations most targeted in Parkinson’s disease. Many cellular redox reactions rely on iron, however an altered distribution of reactive iron is cytotoxic. In fact, increased levels of iron in the brain of Parkinson’s disease patients are present. Dopamine accumulation can induce neuronal death; however, excess dopamine can be removed by converting it into a stable compound like neuromelanin, and this process rescues the cell. Interestingly, the main iron compound in dopamine and norepinephrine neurons is the neuromelanin-iron complex, since neuromelanin is an effective metal chelator. Neuromelanin serves to trap iron and provide neuronal protection from oxidative stress. This equilibrium between iron, dopamine, and neuromelanin is crucial for cell homeostasis and in some cellular circumstances can be disrupted. Indeed, when neuromelanin-containing organelles accumulate high load of toxins and iron during aging a neurodegenerative process can be triggered. In addition, neuromelanin released by degenerating neurons activates microglia and the latter cause neurons death with further release of neuromelanin, then starting a self-propelling mechanism of neuroinflammation and neurodegeneration. Considering the above issues, age-related accumulation of neuromelanin in dopamine neurons shows an interesting link between aging and neurodegeneration.

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The molecular mechanisms causing the loss of dopaminergic neurons containing neuromelanin in the substantia nigra and responsible for motor symptoms of Parkinson's disease are still unknown. The discovery of genes associated with Parkinson's disease (such as alpha synuclein (SNCA), E3 ubiquitin protein ligase (parkin), DJ-1 (PARK7), ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL-1), serine/ threonine-protein kinase (PINK-1), leucine-rich repeat kinase 2 (LRRK2), cation-transporting ATPase 13A1 (ATP13A), etc.) contributed enormously to basic research towards understanding the role of these proteins in the sporadic form of the disease. However, it is generally accepted by the scientific community that mitochondria dysfunction, alpha synuclein aggregation, dysfunction of protein degradation, oxidative stress and neuroinflammation are involved in neurodegeneration. Dopamine oxidation seems to be a complex pathway in which dopamine o-quinone, aminochrome and 5,6-indolequinone are formed. However, both dopamine o-quinone and 5,6-indolequinone are so unstable that is difficult to study and separate their roles in the degenerative process occurring in Parkinson's disease. Dopamine oxidation to dopamine o-quinone, aminochrome and 5,6-indolequinone seems to play an important role in the neurodegenerative processes of Parkinson's disease as aminochrome induces: (i) mitochondria dysfunction, (ii) formation and stabilization of neurotoxic protofibrils of alpha synuclein, (iii) protein degradation dysfunction of both proteasomal and lysosomal systems and (iv) oxidative stress. The neurotoxic effects of aminochrome in dopaminergic neurons can be inhibited by: (i) preventing dopamine oxidation of the transporter that takes up dopamine into monoaminergic vesicles with low pH and dopamine oxidative deamination catalyzed by monoamino oxidase (ii) dopamine o-quinone, aminochrome and 5,6-indolequinone polymerization to neuromelanin and (iii) two-electron reduction of aminochrome catalyzed by DT-diaphorase. Furthermore, dopamine conversion to NM seems to have a dual role, protective and toxic, depending mostly on the cellular context. Dopamine metabolismDopamine synthesis and storage Dopaminergic neurons are involved in motor activity, in which dopamine is synthesized, stored and released to intersynaptic space. Dopamine is synthesized from the amino acid tyrosine in two steps that occur in the cytosol: (i) hydroxylation of tyrosine to l-dihydroxyphenylanaline (l-dopa), a reaction catalyzed by tyrosine hydroxylase (TH) that requires oxygen and (ii) decarboxylation of l-dopa to dopamine, a reaction catalyzed by aromatic amino acid decarboxylase (AADC) that generates CO 2 . Interestingly, dopamine synthesis does not result in dopamine accumulation in the cytosol as a consequence that TH and AADC are associated with the vesicular monoaminergic transporter-2 (VMAT-2) generating a complex where tyrosine is converted to l-dopa that immediately decarboxylated to dopamine. The

Mutations leading to expansion of a poly-glutamine track in Huntingtin (Htt) cause Huntington's disease (HD). Signs of endoplasmic reticulum (ER) stress have been recently reported in animal models of HD, associated with the activation of the unfolded protein response (UPR). Here we have investigated the functional contribution of ER stress to HD by targeting the expression of two main UPR transcription factors, XBP1 and ATF4 (activating transcription factor 4), in full-length mutant Huntingtin (mHtt) transgenic mice. XBP1-deficient mice were more resistant to developing disease features, associated with improved neuronal survival and motor performance, and a drastic decrease in mHtt levels. The protective effects of XBP1 deficiency were associated with enhanced macroautophagy in both cellular and animal models of HD. In contrast, ATF4 deficiency did not alter mHtt levels. Although, XBP1 mRNA splicing was observed in the striatum of HD transgenic brains, no changes in the levels of classical ER stress markers were detected in symptomatic animals. At the mechanistic level, we observed that XBP1 deficiency led to augmented expression of Forkhead box O1 (FoxO1), a key transcription factor regulating autophagy in neurons. In agreement with this finding, ectopic expression of FoxO1 enhanced autophagy and mHtt clearance in vitro. Our results provide strong evidence supporting an involvement of XBP1 in HD pathogenesis probably due to an ER stress-independent mechanism involving the control of FoxO1 and autophagy levels.

o-Quinones are physiological oxidation products of catecholamines that contribute to redox cycling, toxicity and apoptosis, i.e. the neurodegenerative processes underlying Parkinson's disease and schizophrenia. The present study shows that the cyclized o-quinones aminochrome, dopachrome, adrenochrome and noradrenochrome, derived from dopamine, dopa, adrenaline and noradrenaline respectively, are efficiently conjugated with glutathione in the presence of human glutathione transferase (GST) M2-2. The oxidation product of adrenaline, adrenochrome, is less active as a substrate for GST M2-2, and more efficiently conjugated by GST M1-1. Evidence for expression of GST M2-2 in substantia nigra of human brain was obtained by identification of the corresponding PCR product in a cDNA library. Glutathione conjugation of these quinones is a detoxication reaction that prevents redox cycling, thus indicating that GSTs have a cytoprotective role involving elimination of reactive chemical species originating from the oxidative metabolism of catecholamines. In particular, GST M2-2 has the capacity to provide protection relevant to the prevention of neurodegenerative diseases.

The molecular mechanisms involved in the neurodegenerative process of Parkinson's disease remain unclear. Currently, there is a general agreement that mitochondrial dysfunction, α-synuclein aggregation, oxidative stress, neuroinflammation, and impaired protein degradation are involved in the neurodegeneration of dopaminergic neurons containing neuromelanin in Parkinson's disease. Aminochrome has been proposed to play an essential role in the degeneration of dopaminergic neurons containing neuromelanin by inducing mitochondrial dysfunction, oxidative stress, the formation of neurotoxic α-synuclein protofibrils, and impaired protein degradation. Here, we discuss the relationship between the oxidation of dopamine to aminochrome, the precursor of neuromelanin, autophagy dysfunction in dopaminergic neurons containing neuromelanin, and the role of dopamine oxidation to aminochrome in autophagy dysfunction in dopaminergic neurons. Aminochrome induces the following: (i) the formation of α-synuclein protofibrils that inactivate chaperone-mediated autophagy; (ii) the formation of adducts with α- and β-tubulin, which induce the aggregation of the microtubules required for the fusion of autophagy vacuoles and lysosomes.