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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Nerve terminals are unique among cellular secretory systems in that they can sustain vesicular release at a high rate. Although little is known about the mechanisms that account for the distinctive features of neurotransmitter release, it can be assumed that neuron-specific proteins are involved. One such protein family, the synapsins, are believed to regulate neurotransmitter release through phosphorylation-dependent interactions with synaptic vesicles and cytoskeletal elements. Here we show that clusters of vesicles at synaptic release sites are composed of two pools, a distal pool containing synapsin and a proximal pool devoid of synapsin and located adjacent to the presynaptic membrane. Presynaptic injection of synapsin antibodies resulted in the loss of the distal pool, without any apparent effect on the proximal pool. Depletion of this distal pool was associated with a marked depression of neurotransmitter release evoked by high-frequency (18-20 Hz) but not by low-frequency (0.2 Hz) stimulation. Thus the availability of the synapsin-associated pool of vesicles seems to be required to sustain release of neurotransmitter in response to high-frequency bursts of impulses.

One of the crucial issues in understanding neuronal transmission is to define the role(s) of the numerous proteins that are localized within presynaptic terminals and are thought to participate in the regulation of the synaptic vesicle life cycle. Synapsins are a multigene family of neuron-specific phosphoproteins and are the most abundant proteins on synaptic vesicles. Synapsins are able to interact in vitro with lipid and protein components of synaptic vesicles and with various cytoskeletal proteins, including actin. These and other studies have led to a model in which synapsins, by tethering synaptic vesicles to each other and to an actin-based cytoskeletal meshwork, maintain a reserve pool of vesicles in the vicinity of the active zone. Perturbation of synapsin function in a variety of preparations led to a selective disruption of this reserve pool and to an increase in synaptic depression, suggesting that the synapsin-dependent cluster of vesicles is required to sustain release of neurotransmitter in response to high levels of neuronal activity. In a recent study performed at the squid giant synapse, perturbation of synapsin function resulted in a selective disruption of the reserve pool of vesicles and in addition, led to an inhibition and slowing of the kinetics of neurotransmitter release, indicating a second role for synapsins downstream from vesicle docking. These data suggest that synapsins are involved in two distinct reactions which are crucial for exocytosis in presynaptic nerve terminals. This review describes our current understanding of the molecular mechanisms by which synapsins modulate synaptic transmission, while the increasingly well-documented role of the synapsins in synapse formation and stabilization lies beyond the scope of this review.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson’s disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-β (CaMKK-β)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca2+-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

Synapsins, a family of synaptic vesicle proteins, have been shown to regulate neurotransmitter release; the mechanism(s) by which they act are not fully understood. Here we have studied the role of domain E of synapsins in neurotransmitter release at the squid giant synapse. Two squid synapsin isoforms were cloned and found to contain a carboxy (C)-terminal domain homologous to domain E of the vertebrate a-type synapsin isoforms. Presynaptic injection of a peptide fragment of domain E greatly reduced the number of synaptic vesicles in the periphery of the active zone, and increased the rate and extent of synaptic depression, suggesting that domain E is essential for synapsins to regulate a reserve pool of synaptic vesicles. Domain E peptide had no effect on the number of docked synaptic vesicles, yet reversibly inhibited and slowed the kinetics of neurotransmitter release, indicating a second role for synapsins that is more intimately associated with the release process itself. Thus, synapsin domain E is involved in at least two distinct reactions that are crucial for exocytosis in presynaptic terminals.

Synapsins are a family of neuron-specific phosphoproteins that regulate neurotransmitter release by associating with synaptic vesicles. Synapsins consist of a series of conserved and variable structural domains of unknown function. We performed a systematic structurefunction analysis of the various domains of synapsin by assessing the actions of synapsin fragments on neurotransmitter release, presynaptic ultrastructure, and the biochemical interactions of synapsin. Injecting a peptide derived from domain A into the squid giant presynaptic terminal inhibited neurotransmitter release in a phosphorylation-dependent manner. This peptide had no effect on vesicle pool size, synaptic depression, or transmitter release kinetics. In contrast, a peptide fragment from domain C reduced the number of synaptic vesicles in the periphery of the active zone and increased the rate and extent of synaptic depression. This peptide also slowed the kinetics of neurotransmitter release without affecting the number of docked vesicles. The domain C peptide, as well as another peptide from domain E that is known to have identical effects on vesicle pool size and release kinetics, both specifically interfered with the binding of synapsins to actin but not with the binding of synapsins to synaptic vesicles. This suggests that both peptides interfere with release by preventing interactions of synapsins with actin. Thus, interactions of domains C and E with the actin cytoskeleton may allow synapsins to perform two roles in regulating release, whereas domain A has an actin-independent function that regulates transmitter release in a phosphorylation-sensitive manner.