Amyloid β Degradation & Inflammation: New Therapeutic Strategies in AD. Li Gan, Ph.D

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1 ADC Directors Meeting Chicago, April 12, 2008 Amyloid β Degradation & Inflammation: New Therapeutic Strategies in AD Li Gan, Ph.D Gladstone Institute of Neurological Disease University of California, San Francisco

2 Amyloid β Hypothesis in Alzheimer s Disease Aβ Aggregates Degradation Degradation Activated Microglia Insoluble fibrils Soluble oligomers + Plaques Neurodegeneration

3 Amyloid β Hypothesis in Alzheimer s Disease Aβ Aggregates Degradation Activated Microglia Insoluble fibrils Soluble oligomers + Plaques Neurodegeneration

4 Outline 1 Reduce abnormal accumulation of Aβ by promoting degradation 1 2 Block the toxic pathways in microglia activation 2

5 Aβ Accumulation Results From an Imbalance Between Production and Clearance P C

6 Generation of Aβ peptides from Amyloid Precursor Protein (APP) FAD mutations β-secretase (BACE1) Aβ Aβ γ-secretase (Presenilin complex) Aβ42 Aβ40 Aβ38 happ DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA More toxic More prone to aggregation Aβ1-42

7 The Genetics of Alzheimer's Disease Genetic factors are involved in 25 to 40% of AD cases Early-onset Familial AD APP (chromosome 21) PSEN1 (chromosome 14) PSEN2 (chromosome 1) Amyloid β (Aβ42) Modified from Tanzi and Bertram, Cell, 2005

8 The Genetics of Alzheimer's Disease Genetic factors are involved in 25 to 40% of AD cases Early-onset Familial AD APP (chromosome 21) PSEN1 (chromosome 14) PSEN2 (chromosome 1) ~ 5% AD cases Late-onset Sporadic AD APOE (chrosome 19) (ε4 allele confers risk) > 200 Genes ( Modified from Tanzi and Bertram, Cell, 2005

9 Targeting Aβ Accumulation Aβ Production secretase inhibitors Aβ Accumulation Aβ Clearance Degradation Neurodegeneration

10 Targeting Aβ Accumulation Aβ Production secretase inhibitors Aβ Accumulation Neurodegeneration Aβ Clearance Insulin degrading enzyme (IDE) Neprilysin (NEP) Angiotensin converting enzyme (ACE) Endothelin converting enzyme Plasmin MMP-9 Cathepsin B

11 Cathepsin B Is an Cysteine Protease Localized at Amyloid Plaques and Neuronal Endosomes Control AD ontrol happ mice

12 Genetic Ablation of Cathepsin B Increases Aβ Deposition in happ Mice happ/catb +/+ (n=10) happ/catb / (n=12) Anti-Aβ Anti-Aβ Mueller-Steiner et al., Neuron, 51: (2006)

13 Cathepsin B and Neprilysin Gene Transfer Reduces Aβ Deposits in the Dentate Gyrus of happ Mice 3D6 3D6 Anti-Aβ 3D6 3D6 Anti-Aβ Injected-side Injected (Lenti-CatB) Contralateral Non-injected Mueller-Steiner et al., Neuron, 51: (2006)

14 Cathepsin B Degrades Synthetic Aβ1-42 Oligomers Under Cell-free Conditions Aβ1-42 Mueller-Steiner et al., Neuron, 51: (2006)

15 Analysis of CatB-induced Cleavage of Aβ1-42 Monomers Oligomers Negative Staining EM Incubate with purified Cathepsin B Seldi-TOF Mass Spectrometry (Ciphergen Biosystems)

16 Cathepsin B Truncates Aβ1-42 in a Dose- Dependent Manner None Seldi-TOF Mass Spectrometry 100 ng/ml ng/ml 300 ng/ml 400 ng/ml

17 Cystatin C Is an Endogenous Inhibitor of Cathepsin B Cathepsin B Degradation Cystatin C + Neurodegeneration

18 The Genetics of Alzheimer's Disease Early-onset Familial AD APP (21q21) PSEN1 (14q24) PSEN2 (1q42) Late-onset Sporadic AD APOE (19q13) (ε4 allele confers risk) > 200 Genes ( (Meta-analysis confirmed: ACE, CHRNB2, CST3, ESR1, GAPDHS, IDE, MTHFR, NCSTN, PRNP, PSEN1, TF, TFAM and TNF..) Modified from Tanzi and Bertram, Cell, 2005

19 Cystatin C in AD Polymorphism associated with higher risk for lateonset AD Increased in the CSF of AD patients and a subset of neurons in AD-related animal models Inhibit Aβ fibrillization through direct binding to Aβ Kaeser SA et al., & Mi W et al., Nature Genetics, 2007

20 Reducing Cystatin C Will Lower Soluble Aβ by Enhancing CatB Activity CatB CysC

21 Reduction of Cystatin C Elevates the Activity of CatB

22 Genetic Inactivation of Cystatin C in happ Mice X happ + CysC / 2 4-month-old CysC +/+ CysC +/ CysC / 5 8-month-old 8 10-month-old happ/ CysC +/+ happ/cysc +/ happ/cysc /

23 Cystatin C Reduction Lowers Soluble Aβ1-x and Aβ1-42 in Young happ Mice Aβ1-X Aβ month-old

24 β Cystatin C Ablation Lowers the Relative Abundance of Aβ1-42 in Young happ Mice 2 4-month-old

25 Cystatin C Ablation Reduces Plaque Load in happ Mice

26 Ablation of CysC Prevents Calbindin Depletion in the DG of happ mice calbindin CysC +/+ CysC / happ/cysc +/+ happ/cysc /

27 Reduction of CysC Prevents Calbindin Depletion in the DG of happ mice

28 Genotypes Soluble Aβ Aβ1 42/Aβ Plaque Calbindin Fos CST3+/ CST3+/ No human Aβ CST3 / happ/cst3+/ happ/cst3+/ ++ ++* ++* happ/cst3 /

29 Cystatin C Reduction Abolishes Premature Mortality

30 Are the Beneficial Effects of Cystatin C Reduction Mediated Through Enhancing CatB Activity? Cystatin C Cathepsin B Soluble Aβ (42) Plaque load Neuronal Deficits

31 Are the Beneficial Effects of Cystatin C Reduction Mediated Through Enhancing CatB Activity? Cystatin C Binds to Aβ Other cysteine proteinases Cathepsin B Soluble Aβ (42)?? Fibril Aβ Neuronal Deficits

32 Are the Beneficial Effects of Cystatin C Reduction Mediated Through Enhancing CatB Activity? Cystatin C Binds to Aβ Other cysteine proteinases Soluble Aβ?? Fibril Aβ Neuronal Deficits

33 Effects of Cystatin C Reduction in happ Mice Lacking CatB X happ + /CatB / CysC / /CatB / 2 3-month-old CysC +/+ /CatB / CysC +/ /CatB / CysC / /CatB / 4 6-month-old happ/cysc +/+ /CatB / happ/cysc +/ /CatB / happ/cysc / /CatB /

34 CysC Reduction Failed to Lower Soluble Aβ Levels in the Absence of CatB Aβ1-x (ng/g tissue) Aβ1-X Aβ CatB / / / CatB / / / Aβ1-42 (ng/g tissue) month-old happ/cysc+/+ happ/cysc+/ happ/cysc /

35 Reducing CysC Failed to Lower Relative Abundance of Aβ1-42 and Plaque Load in the Absence of CatB 2 3-month-old 4 6-month-old happ/cysc+/+ happ/cysc+/ happ/cysc /

36 CysC Reduction Did Not Increase Calbindin Levels in the Absence of CatB Relative Calbindin Levels happ *** *** *** CysC+/+ CysC+/ CysC / CatB / / / / / /

37 Genotypes Soluble Aβ Aβ1 42/Aβ Plaque Calbindin c-fos CST3+/ CST3+/ No human Aβ CST3 / happ/cst3+/ happ/cst3+/ happ/cst3 / CST3+/+/CatB / CST3+/ /CatB / No human Aβ CST3 / /CatB / happ/cst3+/+/catb / happ/cst3+/ /CatB / happ/cst3 / /CatB /

38 Cystatin C Regulates Soluble Aβ and Neuronal Deficits in a CatB-dependent Manner Cystatin C Binds to Aβ Other cysteine proteinases Cathepsin B Soluble Aβ (42) Aβ Fibrils Neuronal Deficits

39 New Strategies to Reduce Aβ Accumulation Cathepsin B Degradation Cystatin C Gene transfer of CatB enzyme Reducing CysC levels Disrupting the CatB-CysC interaction Neurodegeneration

40 Our Research Focuses 1 Reduce abnormal accumulation of Aβ by promoting degradation 1 2 Block the toxic pathways in microglia activation 2

41 Aβ Downregulates SIRT1 and Activates NF-κB Signaling in Microglia SIRT1 Degradation NF-κB + Neurodegeneration

42 SIRT1 Class III histone deacetylase Deactylate histones and non-histone transcription factors A conserved pathway for lifespan extension from protozoa to metazoa Modulate multiple processes in neurodegeneration Gan and Mucke, Neuron, 58: (2008)

43 Inhibition of NF-κB Signaling Through Deacetylation by SIRT1 Activation of HDAC(SIRT1) HDAC: Histone Deacetylase Activation Adapted from Chen and Greene, J Mol Med., 2003 Yeung et al., 2004

44 Aβ Oligomers Downregulate SIRT1 and Activate NF-κB Activation in Microglial Cells SIRT1 Aβ Tubulin Levels of SIRT1 Relative to Tubulin In BV2 cells CTRL Aβ Aβ Green: NF-κB activation

45 SIRT1 Levels Are Downregulated in happ Mouse Brains

46 Activation of NF-κB Signaling in Hippocampus of happ Mice NF-κB DNA/ NE complex Electrophoretic Mobility Shift Assay (EMSA) Esposito et al. (Mucke), J. Neuroscience

47 Cortical Neuron-Glia Mixed Cultures MAP2 (neurons) GFAP (astrocytes) CD11b (microglia) GFAP (astrocytes)

48 Targeted Inhibition of NF-κB Signaling in Microglia Protects Against Aβ Toxicity Aβ1-42 Aβ1-42 Lenti-MCSF-IκBα-SR MCSF IκBα-SR Chen et al., J. Biol. Chem., 280. (2005)

49 Expression of SIRT1 in Microglia Protects Against Aβ Toxicity Lenti-MCSF-SIRT1 MCSF SIRT1 Adapted from Chen and Greene, J Mol Med., 2003

50 SIRT1 Agonist Resveratrol Inhibits NF-κB and Protects Against Toxic Effects of Microglia Activation NT No treatment Aβ Chen et al., J. Biol. Chem., 280. (2005) Red: MAP2 (neurons) Green: NF-κB activation Aβ+Res

51 Resveratrol Exerts Beneficial Effects as a SIRT1 Agonist in Vivo Grapes Reduces insulin resistance, Increases mitochondrial function, Prolongs survival in mice fed a high-fat diet Red Wine Baur et al., Nature, (2006); Lagouge et al., Cell, (2006)

52 New Strategies to Block the Toxic Effects of Microglia Activation Degradation NF-κB SIRT1 Resveratrol Neurodegeneration Inhibiting NF-κB in microglia Increasing SIRT1 in microglia Addition of SIRT1 agonists

53 Acknowledgments Gan Lab Binggui Sun Yungui Zhou Seo-Hyun Cho Brian Halabisky Sang-Won Min Sarah Mueller-Steiner Jennifer Chen Hideaki Arai Funding National Institute of Aging NIA/UCSF ADRC Pilot Grant Alzheimer s Disease Program of California Whittier Foundation Hellman Family Fund Collaborators Anders Grubb Erik Roberson Xin Wang Lennart Mucke Hidde Plough Christoph Peters Eliezer Masliah

54 SIRT1 NF-κB Aβ Aggregates Degradation Activated Microglia Soluble oligomers + Neurodegeneration

55 Ongoing Investigation 1) Does CysC reduction prevent behavioral deficits in happ mice? Elevated Plus Maze Time of entry in the Open Arm Elevated plus maze Open-field Activity *** ** Morris water maze Contextual fear conditioning CysC happ +/+ / +/+ / + +

56 Relative Total Activity (3rd time/1st time) Ongoing Investigation 1) Does CysC reduction prevent behavioral deficits in happ mice? Habituation in the Open Field P = 0.07 Elevated plus maze Habituation in the open-field Morris water maze Contextual fear conditioning CysC +/+ / +/+ / happ + +

57 Ongoing Investigation & Future Plans 2) Where does CysC and CatB interact? - Extracellular or intracellular - Neuron or glia (microglia) - Autophagic degradation? 3) How is the CysC-CatB axis regulated? 4) How to disrupt the CysC-CatB interaction?

58 Ongoing Investigation & Future Plans 2) Where does CysC and CatB interact? 3) How is the CysC-CatB axis regulated? - Misfolded protein - Aging/environmental factors (stress & inflammation) - Genetic mutations/polymorphism 4) How to disrupt the CysC-CatB interaction to promote Aβ clearance?

59 Intracellular Accumulation of CysC Is Associated With CST3 Polymorphism & PS2 Mutations Mock PS2wt PS2M239I PS2T122R Ghidoni et al., Neurobiology of Aging, 2007 Benussi et al., Neurobiology of Disease, 2003

60 Ongoing Investigation & Future Plans 2) Where does CysC and CatB interact? 3) How is the CysC-CatB axis regulated? 4) How to disrupt the CysC-CatB interaction? - Structural basis - Robust cell-based or cell-free assay - Monoclonal antibodies (extracellular) - Small molecule compounds (Intracellular)

61 Functional Deficits Induced by Naturally Secreted Aβ Oligomers From CHO Cells Expressing happ (7PA2) QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime and a TIFF (Uncompressed) decompressor Trimer are needed to see this picture. Dimer conditioned medium from 7PA2 Cells Walsh et al., Nature, 2002

62 A Cell-based Assay to Screen for Inhibitors of CatB-CysC Interaction Conditioned Medium 7PA2-Mock 7PA2-CysC 7PA2-CysC-Δ Gly-CysC CysC Relative CatB Activity PA2- Mock 7PA2- CysC 7PA2- CysC-Δ

63 A Cell-based Assay to Screen for Inhibitors of CatB-CysC Interaction Multiple readouts: CatB activities (enzymatic assay) Total Aβ levels (ELISA) Levels of Aβ oligomers (IP-WB) Toxicity of the conditioned medium (cell death assay/electrophysiology)

64 Reducing CysC Lowers Relative Abundance of Aβ1-42 in the Presence of CatB happ/cysc+/+ happ/cysc+/ happ/cysc /

65 Relative Abundance of Aβ1-42 Remains Similarly High in the Absence of CatB happ/cysc+/+ happ/cysc+/ happ/cysc /

66 Structural Basis for the CatB-CysC Interaction Regions on CysC that confer selectivity to CatB: - Arg-8 and Leu-9 - Trp106 at the second loop Regions on CatB that interact with CysC: Janowski et al. 2005; Jia et al, 1995; Clare Peters-Libeu

67 Structural Basis for the CatB-CysC Interaction Regions on CysC that confer selectivity to CatB: - Arg-8 and Leu-9 - Trp106 at the second loop Regions on CatB that interact with CysC: Janowski et al. 2005; Jia et al, 1995; Clare Peters-Libeu

68 Cathepsin B Reduces Naturally Secreted Aβ Oligomers in CHO Cells Expressing happ 7PA2 CHO Ct CatB Ct Ct CatB CHO Dimers * 8 8 7PA2 7PA2-CatB CHO 7PA2 Trimers T D M Trimer Dimer 7PA2 CatB * 8 8 CHO 7PA2 7PA2-CatB M. Cisse, Y. Zhou

69 Cystatin C May Interact with Cathepsin B Extracellularly Cathepsin B activity in CSF 2500 Fluorescence Units CysC-/- CysC+/+ Binggui Sun

70 CysC-CatB Axis in Aβ Degradation Co-localization of cystatin C with cathepsin B in neurons of AD brains Deng, et al, Am J Pathol 2001, 59: CysC Aβ Merge Microglial BV2 Cells CatB Aβ Merge

71 Constitutive Autophagy Plays an Central Role in Degradation of Misfolded Proteins Levine & Kroemer, Cell, 2008

72 Macroautophagy Involves Fusion of Lysosomes With Autophagosomes LC3-II LC3-II Levine & Kroemer, Cell, 2008

73 Aβ Activates Autophagy in Microglia: Accompanied by CatB Induction and CysC Reduction Activation of Autophagy LC3-I LC3-II Ctrl LPS Rapamycin Ctrl Ctrl + Aβ oligomers LC3-I LC3-II LC3-I LC3-II Microglial BV2 Cells QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. CatB QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. CysC

74 APP Processing and Aβ Generation α-secretase β-secretase Aβ γ-secretase FL-hAPP A β β-ctf α-ctf α-sapp Aβ1-40 Aβ1-38 Aβ1-42

75 Cystatin C Ablation Does Not Affect happ Processing in Vivo

76 Overexpression of Cystatin C Resulted in Slight Inhibition of CatB Ntg Tg-hCysC Yungui Zhou and Stephan Kaeser

77 DeMattos et al., 2002 Mucke et al., 2000 In the CSF [Ab] ~ 20 ng/ml (4.4 nm) [CysC] ~ 0.1uM =100 nm >> CatB In the hippocampus: [Ab] ~ 50 nm [CysC] ~ 0.1uM =100 nm >> CatB