Limor Avrahami

Bio: Limor Avrahami is an academic researcher from Tel Aviv University. The author has contributed to research in topics: GSK-3 & Autophagy. The author has an hindex of 9, co-authored 11 publications receiving 5248 citations.

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

Abstract: 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. For example, 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 versus those that measure flux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase 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. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. 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. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. 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 assays, we hope to encourage technical innovation in the field.

Combined regulation of mTORC1 and lysosomal acidification by GSK-3 suppresses autophagy and contributes to cancer cell growth

Abstract: There is controversy over the role of glycogen synthase kinase-3 (GSK-3) in cancer progression. Recent work has implicated GSK-3 in the regulation of mammalian target of rapamycin (mTOR), a known player in malignant transformation. Autophagy, a self-degradation pathway, is inhibited by mTOR and is tightly associated with cell survival and tumor growth. Here we show that GSK-3 suppresses autophagy via mTOR complex-1 (mTORC1) and lysosomal regulation. We show that overexpression of GSK-3 isoforms (GSK-3α and GSK-3β) activated mTORC1 and suppressed autophagy in MCF-7 human breast cancer cells as indicated by reduced beclin-1 levels and upregulation of sequestosome 1 (p62/SQSTM1). Further, overexpression of GSK-3 increased the number of autophagosomes and inhibited autophagic flux. This activity was directly related to reduced lysosomal acidification triggered by GSK-3 (in which GSK-3β has a stronger impact). We found that lysosomal acidification is reduced in MCF-7 cells that also exhibit increased levels of autophagosomes and p62/SQSTM1 and increased activity of mTORC1. Subsequently, treating cells with GSK-3 inhibitors restored lysosomal acidification, enhanced autophagic flux and inhibited mTORC1. Furthermore, GSK-3 inhibitors inhibited cell proliferation. We provide evidence that GSK3-mediated mTORC1 activity and GSK-3-mediated lysosomal acidification occur via distinct pathways, yet both mTORC1 and lysosomes control cell growth. Finally, we show that GSK-3-reduced lysosomal acidification inhibits endocytic clearance as demonstrated by reduced endocytic degradation of the epidermal growth factor receptor. Taken together, our study places GSK-3 as a key regulator coordinating cellular homeostasis. GSK-3 inhibitors may be useful in targeting mTORC1 and lysosomal acidification for cancer therapy.

A unique type of GSK-3 inhibitor brings new opportunities to the clinic

Abstract: Development of protein kinase inhibitors is a focus of many drug discovery programs. A major problem, however, is the limited specificity of the commonly used adenosine triphosphate–competitive inhibitors and the weak inhibition of the more selective substrate-competitive inhibitors. Glycogen synthase kinase–3 (GSK-3) is a promising drug target for treating neurodegenerative disorders, including Alzheimer’s disease (AD), but most GSK-3 inhibitors have not reached the clinic. We describe a new type of GSK-3 inhibitor, L807mts, that acts through a substrate-to-inhibitor conversion mechanism that occurs within the catalytic site of the enzyme. We determined that L807mts was a potent and highly selective GSK-3 inhibitor with reasonable pharmacological and safety properties when tested in rodents. Treatment with L807mts enhanced the clearance of β-amyloid loads, reduced inflammation, enhanced autophagic flux, and improved cognitive and social skills in the 5XFAD AD mouse model. This new modality of GSK-3 inhibition may be therapeutic in patients with AD or other central nervous system disorders associated with dysregulated GSK-3.

Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.

Abstract: Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

Abstract: In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including 'apoptosis', 'necrosis' and 'mitotic catastrophe'. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

The role of autophagy in neurodegenerative disease

Abstract: This Review provides an overview of the role of autophagy, a key lysosomal degradative process, in neurodegenerative diseases. The study of various neurodegenerative diseases has shown that defects in autophagy can arise at different points in the pathway, and this has implications for the successful modulation of autophagy for therapeutic purposes. The Review also discusses the latest developments in targeting alterations in autophagy as a therapeutic strategy for neurodegenerative diseases.

Targeting autophagy in cancer

Abstract: Autophagy is a mechanism by which cellular material is delivered to lysosomes for degradation, leading to the basal turnover of cell components and providing energy and macromolecular precursors. Autophagy has opposing, context-dependent roles in cancer, and interventions to both stimulate and inhibit autophagy have been proposed as cancer therapies. This has led to the therapeutic targeting of autophagy in cancer to be sometimes viewed as controversial. In this Review, we suggest a way forwards for the effective targeting of autophagy by understanding the context-dependent roles of autophagy and by capitalizing on modern approaches to clinical trial design.