ALZHEIMER S DISEASE (AD)

Transcription

1 ALZHEIMER S DISEASE (AD) AD IS COMMON, PROGRESSIVE, AND INCURABLE ONLY A FEW TREATMENTS KNOWN-HAVE MINIMAL EFFECT SYNAPSE LOSS EVENTUAL MASSIVE NEURONAL DEATH PLAQUES THE AMYLOID CASCADE HYPOTHESIS TANGLES FROM WWW- MEDLIB.MED.UTAH.EDU/WEBPATH/WEB PATH.HTML APP BETA-SECRETASE (BACE) EXTRACELLULAR DOMAIN TRANSMEMBRANE A FROM y/binder_l/ GAMMA-SECRETASE (PRESENILIN) CYTOPLASMIC 1

2 ARE THERE PROBLEMS WITH THE AMYLOID CASCADE HYPOTHESIS? LACK OF A CREDIBLE MOLECULAR MECHANISM MODEST AMYLOID TOXICITY IN ANIMAL MODELS IN VIVO CONSIDERABLE CORRELATIVE EVIDENCE BUT LITTLE DIRECT EVIDENCE A MOVING TARGET FAD MUTANTS CAUSE ELEVATED AMYLOID (A 42), BUT ALSO MAY HAVE OTHER NON-A 42 DEPENDENT PHENOTYPES INCONSISTENT SUPPORTING DATA IN LITERATURE 2

3 MAKING PROGRESS ON ALZHEIMERS DISEASE (AD) THERE ARE MANY COMPETING AD HYPOTHESES BASED PRIMARILY ON EXPERIMENTS WITH GENETICALLY MODIFIED (OVEREXPRESSION) ANIMALS SUCH ANIMALS ONLY DEVELOP SOME SYMPTOMS OF ALZHEIMER S DISEASE HOW CAN BASIC BIOLOGY BE LINKED TO AD NEURONAL MISBEHAVIOR? WHAT IS RELATIVE TOXICITY TO HUMAN NEURONS OF A VS. APP COMPETITION VS.??? HOW CAN THESE HYPOTHESES BE TESTED? HOW CAN BETTER DRUGS BE TESTED AND DEVELOPED? 3

4 HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSC) WHY BOTHER? CAN MAKE PURE OR DEFINED MIXTURES OF NEURONS AND ASTROCYTES WITH UNIQUE HUMAN BIOCHEMISTRY IN CULTURE EUPLOID AND GENETICALLY STABLE CAPTURE RANGE OF HUMAN GENOMIC VARIATION CAN INDUCE OR CAPTURE FAD OR OTHER POINT MUTATIONS THAT EXPRESS AT CORRECT LEVELS GOOD QUALITY ELECTROPHYSIOLOGY USEFUL FOR BASIC SCIENCE AND DISEASE MODELING AND DRUG SCREENING FAD NEURONS EXHIBIT USEFUL AD PHENOTYPES CAN EVALUATE RISK VARIANTS IN NORMAL CONTEXT HIGHLY MANIPULABLE CAN BE TRANSPLANTED INTO USEFUL ANIMAL MODELS TECHNOLOGY IMPROVING RAPIDLY 4

5 DEVELOPING HUMAN FAMILIAL AD MODELS RARE FORMS OF ALZHEIMER S DISEASE ARE FAMILIAL =FAD A FEW KEY GENES KNOWN MAKING IPS CELLS FROM FAD MUTANT FIBROBLASTS PS1-SHAUNA YUAN, GRACE WOODRUFF, JESSICA YOUNG, AND FERNANDO MARTINEZ APP POINT V717F-GRACE WOODRUFF APP DUPLICATION-MASON ISRAEL DO FAD IPS CELLS DEVELOP ALL AD PHENOTYPES IN CULTURE? CAN THEY BE USED TO TEST HYPOTHESES AND SCREEN FOR DRUGS? 5

6 DEVELOPING SPORADIC HUMAN GENETIC MODELS MOST AD IS SPORADIC=SAD MULTIPLE SAD IPS LINES AND CONTROLS GENERATED-MASON ISRAEL DO SAD IPS CELLS DEVELOP ANY OR ALL AD PHENOTYPES IN CULTURE? CAN THEY BE USED TO TEST HYPOTHESES AND SCREEN FOR DRUGS? 6

7 FACS PURIFICATION OF NEURONS AND GLIA CHRISTIAN CARSON-BD SHAUNA YUAN JESSICA FLIPPIN YUAN ET AL.,

8 SORTED NEURONS YUAN ET AL., PLOS ONE 2011 ISRAEL ET AL., NATURE

9 GENERAL SCHEME FOR ANALYSIS OF FAD AND SAD HIPSC LINES ISRAEL ET AL., NATURE

10 AD PHENOTYPES IN FAD AND SAD HIPSC DERIVED NEURONS ISRAEL ET AL., NATURE 2012 A PEPTIDE? GSK3 KINASE? PHOSPHO- TAU? NDC SAD FAD NDC SAD FAD NDC SAD FAD 10

11 TESTING ROLE OF APP PROCESSING IN INDUCTION OF HYPERPHOSPHORYLATED TAU AND ACTIVATED GSK3 TRANSMEMBRANE APP BETA-SECRETASE (BACE) EXTRACELLULAR DOMAIN A GAMMA-SECRETASE (PRESENILIN) CYTOPLASMIC ISRAEL ET AL., NATURE

12 WHAT DO WE THINK THESE DATA ARE TELLING US? PURIFIED NEURONS FROM FAD PATIENTS RECAPITULATE EARLY FEATURES OF AD BIOCHEMISTRY APP PROCESSING, BUT NOT ABETA DRIVES DOWNSTREAM BIOCHEMICAL PHENOTYPES IN PURIFIED NEURONS CARRYING AN APP DUPLICATION ONE SAD GENOME GENERATES FAD-LIKE PHENOTYPES-MIGHT THIS PROVIDE ASSAY FOR RISK ESTIMATES OR EARLY DIAGNOSIS? PURIFIED AD NEURONS ALLOW MECHANISMS AND DRUGS TO BE TESTED 12

13 IMPORTANT BOTTLENECKS MIXED NEURONAL POPULATIONS PURE NEURONS SO FAR LABORIOUS METHODS FOR HIPSC GENERATION AND NEURONAL DIFFERENTIATION/PURIFICATION GENETIC BACKGROUNDS CONTROLLING VARIABILITY 13

14 DISSECTING HUMAN GENETIC VARIATION DISEASE SUSCEPTIBILITY RESPONSE TO DRUGS (PHARMACOGENOMICS) BEYOND GENOMES THE CRAIG VENTER PROJECT 14

15 REPROGRAMMING CRAIG VENTER EGG SKIN CELL (OOCYTE) ONLY MAKES (REMOVE OOCYTE NUCLEUS) SKIN INNER CELL MASS (PLURIPOTENT) NS FUSION SOMATIC CELL NUCLEAR TRANSFER BLASTOCYST PERSON WITH SPORADIC OR HEREDITARY DISEASE INNER CELL MASS CULTURED PLURIPOTENT STEM CELLS MASON ISRAEL JESSICA YOUNG ISABEL CANTO MELISSA WILBERT ADAPTED FROM 15

16 REPROGRAMMING CRAIG VENTER EGG SKIN CELL (OOCYTE) ONLY MAKES (REMOVE OOCYTE NUCLEUS) SKIN INNER CELL MASS (PLURIPOTENT) NS FUSION SOMATIC CELL NUCLEAR TRANSFER BLASTOCYST INNER CELL MASS CULTURED PLURIPOTENT STEM CELLS MASON ISRAEL JESSICA YOUNG ISABEL CANTO MELISSA WILBERT ADAPTED FROM 16

17 REPROGRAMMING CRAIG VENTER EGG SKIN CELL (OOCYTE) ONLY MAKES (REMOVE OOCYTE NUCLEUS) SKIN INNER CELL MASS (PLURIPOTENT) NS FUSION SOMATIC CELL NUCLEAR TRANSFER BLASTOCYST INNER CELL MASS CULTURED PLURIPOTENT STEM CELLS MASON ISRAEL JESSICA YOUNG ISABEL CANTO MELISSA WILBERT ADAPTED FROM 17

18 REPROGRAMMING CRAIG VENTER EGG SKIN CELL (OOCYTE) ONLY MAKES (REMOVE OOCYTE NUCLEUS) SKIN INNER CELL MASS (PLURIPOTENT) NS FUSION SOMATIC CELL NUCLEAR TRANSFER BLASTOCYST INNER CELL MASS CULTURED PLURIPOTENT STEM CELLS MASON ISRAEL JESSICA YOUNG ISABEL CANTO MELISSA WILBERT ADAPTED FROM 18

19 PROBING SAD RISK VARIANTS Common Biological Pathways: Cholesterol Metabolism Vesicle-mediated transport and endocytosis Immune Function Oligati,

20 WHAT IS SORL1/SORLA? Hemey, G ENDOSOMAL SORTING FACTOR FOR APP COMMON ALLELES/HAPLOTYPES INCREASE SAD RISK MORE SORL1 LEADS TO REDUCED BAD APP PROCESSING AND A PRODUCTION LESS SORL1 LEADS TO INCREASED BAD APP PROCESSING AND A PRODUCTION PREVAILING HYPOTHESIS: COMMON SORL1 RISK VARIANTS DECREASE SORL1 EXPRESSION 20

21 DO COMMON SORL1 RISK VARIANTS DECREASE BASAL EXPRESSION LEVELS? Relative Levels Relative Levels SORL1 EXPRESSION IN FIBROBLASTS, NSC, AND NEURONS: NO OBVIOUS CORRELATION WITH RISK VS. PROTECTIVE ALLELE!! SORL1 mrna: FIBROBLASTS SORL1 mrna: NSCs 2 SORL1 mrna: Neurons P/P R/P R/R P/P R/P R/R P=PROTECTIVE R=RISK JESSICA YOUNG 21

22 SORL1/TBP: Relative Expression SORL1 EXPRESSION SORL1 EXPRESSION INCREASES DURING NEURAL DIFFERENTIATION FROM HIPSC LINES, BUT ABSOLUTE LEVELS ARE HIGHLY VARIABLE SORL1 INDUCTION REDUCED IN CELL LINES FROM R/R PATIENTS SORL1 mrna in neuronal stem cells * * +++ is: +BDNF +GDNF +dbcamp 1 0 NT NT NT P/P R/P R/R # patients = P=PROTECTIVE R=RISK JESSICA YOUNG 22

23 Fold Change SORL1 EXPRESSION SORL1 EXPRESSION INCREASES DURING NEURAL DIFFERENTIATION FROM HIPSC LINES SORL1 INDUCTION REDUCED IN CELL LINES FROM R/R PATIENTS SORL1 mrna induction in purified neurons NT +BDNF NT +BDNF NT +BDNF P/P R/P R/R #patients = P=PROTECTIVE R=RISK JESSICA YOUNG 23

24 DOES BDNF INDUCTION OF SORL1 EXPRESSION CORRELATE WITH REDUCED ABETA PRODUCTION? Fold Change 1.2 Amyloid Beta: NT +BDNF NT +BDNF NT +BDNF P/P R/P R/R #patients = JESSICA YOUNG YES, BUT KEY QUESTION-HOW MUCH OF ABETA REDUCTION CAN BE LINKED TO SORL1? 24

25 HOW MUCH OF ABETA REDUCTION CAN BE LINKED TO SORL1 INCREASE? JESSICA YOUNG 25

26 CURRENT SUMMARY SORL1 RISK VARIANTS MAY REDUCE INDUCTION OF EXPRESSION IMPACT OF SORL1 VARIANT VARIES AMONG BACKGROUNDS INCREASED SORL1 MAY LEAD TO REDUCED A DRUGS TO ACTIVATE PATHWAY? STRATIFICATION OF CLINICAL TRIALS BY SORL1 GENOTYPE? 26

27 THE SOCIOLOGY PROBLEM 27

28 THE SOCIOLOGY PROBLEM FROM AN NIH STUDY SECTION: The use of ips neurons is an advantage when compared to cultured immortalized cell lines or mouse primary neurons. However, they are not superior to animal models of the disease, which have been engineered to over-express different fragments of the APP molecule and/or different human genes that are linked to AD. Relevant to this proposal is the fact that mouse models over-expressing only human Ab, only human CTF/AICD, or the entire human APP, which would be valuable to test the cell non-autonomous, cell-autonomous, or the two-hit models proposed by the PI, already exist. The experiments delineated in this proposal are also limited by the fact that they do not test novel hypothesis, are not designed to provide new information, and are reminiscent of identical studies performed with mouse primary neurons. -AND- Weaknesses The proposed cell non-autonomous, cell-nonautonomous, or the two-hit models could be tested with already available mouse models of the disease. The experiments delineated in this proposal are limited by the fact that they do not test novel hypothesis, are not designed to provide new information, and are reminiscent of identical studies performed with mouse primary neurons. FROM NATURE MEDICINE: In this case, the experts we consulted requested insight into the molecular mechanism as to how loss of NPC affects autophagy in the way you describe, and felt that, for Nature Medicine, it would also be quite important to demonstrate the relevance of the findings to disease pathogenesis in vivo in the animal model. 28

29 HUMAN MODELS OF AD MASON ISRAEL-UCSD SHAUNA YUAN-UCSD SORTING COLLABORATORS SOL REYNA-UCSD CHRISTIAN CARSON-BD CHERYL HERRERA-UCSD JODY MARTIN-BD JESSICA YOUNG-UCSD JEANNE ELIA-BD CEDRIC BARDY-SALK ROSANTO PARAMBAN-BD YANGLING MU-SALK JASON VIDAL-BD FRED GAGE-SALK YANGLING MU-SALK FRED GAGE-SALK JAN STRNADEL-UCSD SILVIA MARSALA-UCSD AD COLLABORATORS MARTIN MARSALA-UCSD BEN YU & UCSD ADRC (SKIN BIOPSIES) ANNE REMES (FINNISH FIBROBLASTS) EDDIE KOO STEVE DOWDY LAB (REPROGRAMMING VECTORS) BIOPSY PARTICIPANTS! 29