Ana Maria Cuervo MD PhD Department of Anatomy and Structural Biology Marion Bessin Liver Research Center Albert Einstein College of Medicine, Bronx, NY Reactivating Chaperone-mediated Autophagy: the advantages of preserving a selective autophagy

Protein damage/repair Aggregates Young Protein Insult Chaperones Repaired Proteases Amino acids Protein Old Chaperones Proteases cv ATP Free radicals O. O. O.

 Overview of CMA  CMA and Aging  Changes  Consequences  Causes  Restorative efforts  Overview of CMA Chaperone-mediated Autophagy and Aging

Types of Autophagy in Mammals lysosome Golgi endoplasmic reticulum MACROAUTOPHAGY ENDOCYTOSIS autophagic vacuole endosome MICROAUTOPHAGY lysosome CHAPERONE- MEDIATED AUTOPHAGY

Chaperone-Mediated Autophagy (CMA) substrate proteins KFERQ-motif lysosome membrane receptor lamp2a proteases lys-hsc70 cytosolic chaperone(s) hsc70/cochaperones

Types of Autophagy Chaperone-mediatedMacroautophagyMicroautophagy ConstitutiveInducible Vesicle-mediated Direct transport Proteins/organelles Proteins NonselectiveNonselective?Selective

Characteristics of Chaperone- Mediated Autophagy  Described: Fibroblasts in culture, other cells type Animal tissues (liver, kidney, spleen)  Regulation: Nutrient deprivation (Stress) Toxic exposure Oxidative stress  Substrates: Selective cytosolic proteins (30%)  Target signal: KFERQ-like  Malfunctioning: Toxic-induced nephropathy Galactosialidosis Aging Parkinson’s Disease Hsp90 Hop Hsp40 LysHsc70 Bag-1 Hip KFERQ-motif substrate CYTOSOL LUMEN Lamp2a LysHsc70

 Overview of CMA  CMA and Aging  Changes  Consequences  Causes  Restorative efforts  CMA and Aging Chaperone-mediated Autophagy and Aging

CMA in old Fibroblasts [ 3 H]Leu Fibroblast - Serum + Serum [ 3 H]amino acids DEGRADATION OF PROTEINS IN FIBROBLASTS Okada & Dice, 1984 Serum TIME (h) DPM IN CELLS (%) Serum + YOUNG FIBROBLASTS 15 OLD FIBROBASTS TIME (h) DPM IN CELLS (%) Serum + 15 Serum -

Degradation of CMA Substrates by Lysosomes Proteolysis (%) YOUNG OLD GAPDH OLD (22m) ** YOUNG (3m) GAPDH Proteolysis (%) Intact lysosomesBroken lysosomes Cuervo & Dice (2000) J. Biol. Chem.

 Overview of CMA  CMA and Aging  Changes  Consequences  Causes  Restorative efforts Chaperone-mediated Autophagy and Aging  Consequences

Protein Degradation and Aging  Intracellular Protein Content Increases with Age  Accumulation of Damaged Proteins is a Common Feature of Old Tissues Does CMA participate in damaged protein removal?

Proteolytic systems inside the cell Nuclei Cytosol Proteasome Lysosome MICROAUTOPHAGY MACROAUTOPHAGY CMA

Oxidized proteins in lysosomes Cytosol Anti-DNPH PQ - + Cytosol PQ In vivo paraquat 24h Saline serum Lysosomes Membrane Matrix - + Liver Mitochondria/ lysosomes Lysosomes Hypotonic shock Membrane Matrix 37 °C - + Washed Membranes NaCl Incubated Matrix - + Roberta Kiffin Kiffin et al. (2004) Mol. Biol. Cell

Lysosome paraquat 24h saline Lysosome Substrate CMA During Mild Oxidative Stress Broken lysosomes Intact lysosomes Proteolysis (%) Cyt Ctr Cyt H 2 O 2 * Lys Ctr GAPDH Proteolysis (%) RNase A Lys PQ *** **

CMA and Oxidative Stress lysosome proteases lamp2a substrate chaperones Hsp90 Hop Hsp40 LysHsc70 Bag-1 Hip LUMEN Lamp2a LysHsc70 L. Membr lamp2a lamp Fed Strv PQ

Consequences of declined CMA with age Impaired elimination of oxidized proteins _

Consequences of CMA blockage lysosome lys-hsc70 substrate chaperones lamp2a Lysosome MACROAUTOPHAGY CMA Ashish Massey Massey et al. (submitted) Lamp2a(-) c1 Lamp2a(-) c2 CMA blockage (fibroblasts RNAi lamp2a)

Effect of CMA Blockage CHAPERONE MEDIATED AUTOPHAGY lysosome WILD TYPE Selective Low capacity MACROAUTOPHAGY autophagic vacuole CMA (-) Nonselective High capacity Are cells OK with this switch?

Consequences of CMA blockage Massey et al. (submitted) Deregulation of the response to stress MTT- Viability assay 24 hours post-insult H (50  M) Paraquat (1 mM) Cadmium (10  M) 42 °C Viable cells (%) wt CMA (-) UV CMA (-) Apoptosis wt noneH2O2H2O2 UV42 °Cpq AnnV(+) 7ADD(-) (% cells))

Consequences of declined CMA with age Impaired elimination of oxidized proteins Deregulation of the response to stress _

 Overview of CMA  CMA and Aging  Changes  Consequences  Causes  Restorative efforts Chaperone-mediated Autophagy and Aging  Causes

CMA and Aging: Step by Step lysosome proteases lys-hsc70 OK substrate chaperones lamp2a 3 m 22 m 3 m 22 m AGE (months) Cuervo and Dice (2000) J. Biol. Chem.

lysosome Normal Conditions protease Nutrient Deprivation lamp2a Substrate Regulation of CMA Redistribution Degradation OK

Lysosomal membrane Lysosomal matrix Lysosomal membrane Proteomics of the Lysosomal Membrane Guy Sovak NaClNa 2 CO 3 Detergent Rat liver lysosomal membranes Lysosomes Lys. membrane 3 m22 m22m CR

 Overview of CMA  CMA and Aging  Changes  Consequences  Causes  Restorative efforts Chaperone-mediated Autophagy and Aging Can we repair the deffect? proteases lys-hsc70 OK lamp2a 3 m 22 m 3 m 22 m AGE (months)

Caloric Restriction and CMA CR 22 22CR Age lamp2a lamp lamp2s 22m CR 12m CR Caloric Restricted 14 GAPDH degradation m12m22m Proteolysis(%) Add Libitum Anna Kim

PALB tetRVP16 tTA The “tet-off” lamp2a mouse + Dox Lamp2a The CMA-Regulated Animal Model lamp2a tet0 P hCMV*-1 - dox+ dox lamp2a tet0 P hCMV*-1 - Doxycycline Lamp2a (+) WT Lamp2a Lamp1 Lamp2a (+) WT + Doxycycline Months Levels lamp2a Dox “Judy” Zhang

The CMA-Regulated Animal Model Cong Zhang Months Levels lamp2a Dox TL2a (tet-) 22monts TL2a (tet+) 22months 10 Cytosol (Oxyblot) WT 6months WT 22months MW IP

1 month Pool Lysosomes CMA+ Lysosomes CMA- Lysosomes 3 months TL2a Activation WT Months Levels lamp2a Dox Late restoration Oxyblot-2D cytosol 26 months WT TL2A-2 1 month activation TL2A-1 1 month activation TL2A-3 3months activation

There is hope for CMA…… …is that all?

AGING Autophagy and Aging Chaperone-mediatedMacroautophagyMicroautophagy?? Lamp2a YoungOld autophagosome autophagolysosome Lysosome ? E. Bergamini Glucagon

Autophagy Crosstalking Lysosome MACROAUTOPHAGY CMA Susmita Kaushik wtATG5(-/-) BROKEN ATG5(-/-) INTACT Proteolysis (%) wt WT

Proteolytic cross-talking Lysosome MICROAUTOPHAGY MACROAUTOPHAGY CMA Proteasome TISSUETIME

The Faces of CMA lysosome CMA and Neurodegeneration Marta Martinez Oxidative Stress Roberta Kiffin Blockage of CMA Ashish Massey Lamp2a Transgenic Mouse Judy Zhang Identification New components Urmi Bandyopadhyay Lysosome Proteome and Aging Guy Sovak Susmita Kaushik Cross-talking Anna Kim CR

Acknowledgements J. Fred Dice (Tufts University, MA) David Sulzer, Serge Przedborski (Columbia U., NY) Peter Lansbury (Harvard University, MA) Harry Ischiropoulos (U. Penn, PA) Ralph Nixon (New York University, NY) Noburu Mizushima (Tokyo MI, Japan) COLLABORATORS CMA CMA and PD FUNDING NIH/NIA, NIH/NIDK Howard Hughes Biomedical Institute Ellison Medical Foundation Huntington’s Disease Society of America Autophagy and AD Cross-talking