Susmita Kaushik

University of Michigan, Ann Arbor, MI, United States

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Publications (46)392.03 Total impact

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    ABSTRACT: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is 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 verify an autophagic response.
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    Dataset: Bunbury 09
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    Dataset: Dev Cell
  • Susmita Kaushik, Ana Maria Cuervo
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    ABSTRACT: Cells continuously turn over proteins through cycles of synthesis and degradation in order to maintain a functional proteome and to exert a tight control in the levels of regulatory proteins. Selective degradation of proteins was initially thought to be an exclusive function of the ubiquitin-proteasome system, however, over the years, the contribution of lysosomes to this selective degradation, through the process of autophagy, has become consolidated. In this context, molecular chaperones, classically associated with protein folding, unfolding and assembling have been revealed as important modulators of selectivity during the autophagic process. Here, we review this relatively new role of chaperones in mediating selective autophagy and comment on how alterations of this function can lead to human pathologies associated to proteotoxicity.
    Pharmacological Research 10/2012; · 4.35 Impact Factor
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    ABSTRACT: A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.
    Cell Reports 07/2012; 2(1):136-49. · 7.21 Impact Factor
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    ABSTRACT: A hallmark of aging is an imbalance between production and clearance of reactive oxygen species and increased levels of oxidatively damaged biomolecules. Herein, we demonstrate that splenic and nodal antigen-presenting cells purified from aging mice accumulate oxidatively modified proteins with side-chain carbonylation, advanced glycation end products, and lipid peroxidation. Furthermore, we show that the endosomal accumulation of oxidatively modified proteins interferes with the efficient processing of exogenous antigens and degradation of macroautophagy-delivered proteins. In support of a causative role for oxidized products in the inefficient immune response, a decrease in oxidative stress improved the adaptive immune response to immunizing antigens. These findings underscore a previously unrecognized negative effect of age-dependent changes in cellular proteostasis on the immune response.
    Cell. 07/2012;
  • Susmita Kaushik, Ana Maria Cuervo
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    ABSTRACT: All cellular proteins undergo continuous synthesis and degradation. This permanent renewal is necessary to maintain a functional proteome and to allow rapid changes in levels of specific proteins with regulatory purposes. Although for a long time lysosomes were considered unable to contribute to the selective degradation of individual proteins, the discovery of chaperone-mediated autophagy (CMA) changed this notion. Here, we review the characteristics that set CMA apart from other types of lysosomal degradation and the subset of molecules that confer cells the capability to identify individual cytosolic proteins and direct them across the lysosomal membrane for degradation.
    Trends in cell biology 06/2012; 22(8):407-17. · 12.12 Impact Factor
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    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. 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.
    Autophagy 04/2012; 8(4):445. · 12.04 Impact Factor
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    Autophagy 04/2012; 8(4):1-100. · 12.04 Impact Factor
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    [Show abstract] [Hide abstract]
    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. 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);5,6 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.
    Autophagy 04/2012; 8(4). · 12.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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. 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.
    Autophagy 04/2012; 8(4):445-544. · 12.04 Impact Factor
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    ABSTRACT: Autophagy degrades cytoplasmic contents to achieve cellular homeostasis. We show that selective loss of autophagy in hypothalamic proopiomelanocortin (POMC) neurons decreases α-melanocyte-stimulating hormone (MSH) levels, promoting adiposity, impairing lipolysis and altering glucose homeostasis. Ageing reduces hypothalamic autophagy and α-MSH levels, and aged-mice phenocopy, the adiposity and lipolytic defect observed in POMC neuron autophagy-null mice. Intraperitoneal isoproterenol restores lipolysis in both models, demonstrating normal adipocyte catecholamine responsiveness. We propose that an unconventional, autophagosome-mediated form of secretion in POMC neurons controls energy balance by regulating α-MSH production. Modulating hypothalamic autophagy might have implications for preventing obesity and metabolic syndrome of ageing.
    EMBO Reports 03/2012; 13(3):258-65. · 7.19 Impact Factor
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    ABSTRACT: Cytosolic proteins can be selectively delivered to lysosomes for degradation through a type of autophagy known as chaperone-mediated autophagy (CMA). CMA contributes to intracellular quality control and to the cellular response to stress. Compromised CMA has been described in aging and in different age-related disorders. CMA substrates cross the lysosomal membrane through a translocation complex; consequently, changes in the properties of the lysosomal membrane should have a marked impact on CMA activity. In this work, we have analyzed the impact that dietary intake of lipids has on CMA activity. We have found that chronic exposure to a high-fat diet or acute exposure to a cholesterol-enriched diet both have an inhibitory effect on CMA. Lysosomes from livers of lipid-challenged mice had a marked decrease in the levels of the CMA receptor, the lysosome-associated membrane protein type 2A, because of loss of its stability at the lysosomal membrane. This accelerated degradation of lysosome-associated membrane protein type 2A, also described as the mechanism that determines the decline in CMA activity with age, results from its increased mobilization to specific lipid regions at the lysosomal membrane. Comparative lipidomic analyses revealed qualitative and quantitative changes in the lipid composition of the lysosomal membrane of the lipid-challenged animals that resemble those observed with age. Our findings identify a previously unknown negative impact of high dietary lipid intake on CMA and underscore the importance of diet composition on CMA malfunction in aging.
    Proceedings of the National Academy of Sciences 02/2012; 109(12):E705-14. · 9.81 Impact Factor
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    ABSTRACT: Autophagy contributes to the removal of prone-to-aggregate proteins, but in several instances these pathogenic proteins have been shown to interfere with autophagic activity. In the case of Huntington's disease (HD), a congenital neurodegenerative disorder resulting from mutation in the huntingtin protein, we have previously described that the mutant protein interferes with the ability of autophagic vacuoles to recognize cytosolic cargo. Growing evidence supports the existence of cross talk among autophagic pathways, suggesting the possibility of functional compensation when one of them is compromised. In this study, we have identified a compensatory upregulation of chaperone-mediated autophagy (CMA) in different cellular and mouse models of HD. Components of CMA, namely the lysosome-associated membrane protein type 2A (LAMP-2A) and lysosomal-hsc70, are markedly increased in HD models. The increase in LAMP-2A is achieved through both an increase in the stability of this protein at the lysosomal membrane and transcriptional upregulation of this splice variant of the lamp-2 gene. We propose that CMA activity increases in response to macroautophagic dysfunction in the early stages of HD, but that the efficiency of this compensatory mechanism may decrease with age and so contribute to cellular failure and the onset of pathological manifestations.
    Journal of Neuroscience 12/2011; 31(50):18492-505. · 6.91 Impact Factor
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    ABSTRACT: Macroautophagy is a lysosomal degradative pathway that maintains cellular homeostasis by turning over cellular components. Here we demonstrate a role for autophagy in hypothalamic agouti-related peptide (AgRP) neurons in the regulation of food intake and energy balance. We show that starvation-induced hypothalamic autophagy mobilizes neuron-intrinsic lipids to generate endogenous free fatty acids, which in turn regulate AgRP levels. The functional consequences of inhibiting autophagy are the failure to upregulate AgRP in response to starvation, and constitutive increases in hypothalamic levels of pro-opiomelanocortin and its cleavage product α-melanocyte-stimulating hormone that typically contribute to a lean phenotype. We propose a conceptual framework for considering how autophagy-regulated lipid metabolism within hypothalamic neurons may modulate neuropeptide levels to have immediate effects on food intake, as well as long-term effects on energy homeostasis. Regulation of hypothalamic autophagy could become an effective intervention in conditions such as obesity and the metabolic syndrome.
    Cell metabolism 08/2011; 14(2):173-83. · 17.35 Impact Factor
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    ABSTRACT: The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.
    Autophagy 07/2011; 7(7):788-9. · 12.04 Impact Factor
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    ABSTRACT: Autophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVBs). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways.
    Developmental Cell 03/2011; 20(3):405-6. · 12.86 Impact Factor
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    Journal of Cell Science 02/2011; 124(Pt 4):495-9. · 5.88 Impact Factor
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    ABSTRACT: Abstract Autophagy delivers cytosolic components to lysosomes for their degradation. The delivery of autophagic cargo to late endosomes for complete or partial degradation has also been described. In this report we present evidence that distinct autophagic mechanisms control cytosolic protein delivery to late endosomes and identify a microautophagy-like process that delivers soluble cytosolic proteins to the vesicles of late endosomes/multivesicular bodies (MVBs). This microautophagy-like process has selectivity and is distinct from chaperone-mediated autophagy that occurs in lysosomes. Endosomal microautophagy occurs during MVB formation, relying on the ESCRT I and III systems for formation of the vesicles in which the cytosolic cargo is internalized. Protein cargo selection is mediated by the chaperone hsc70 and requires the cationic domain of hsc70 for electrostatic interactions with the endosomal membrane. Therefore, we propose that endosomal microautophagy shares molecular components with both the endocytic and autophagic pathways.
    Dev Cell. 01/2011;

Publication Stats

3k Citations
392.03 Total Impact Points

Institutions

  • 2012
    • University of Michigan
      • Life Sciences Institute
      Ann Arbor, MI, United States
  • 2006–2012
    • Albert Einstein College of Medicine
      • • Department of Medicine
      • • Department of Developmental and Molecular Biology
      • • Department of Anatomy and Structural Biology
      New York City, NY, United States
  • 2008
    • National Cancer Institute (USA)
      • Laboratory of Human Carcinogenesis
      Maryland, United States
    • Yeshiva University
      • Department of Anatomy and Structural Biology
      New York City, New York, United States