1. GENE EXPRESSION CHANGES TRIGGERED BY AMYLOID BETA TOXICITY
WAIL M. HASSAN
UNIVERSITY OF WISCONSIN - MILWAUKEE

2. OVERVIEW Model organism Gene expression alterations induced by Aβ
Aβ-specific gene alterations
Membrane damage
Proteasome modulators

3. WHY STUDY GLOBAL GENE EXPRESSION IN NEMATODES?
Ease of genetic manipulations
Short lifespan
Common pathways and orthologues
Inducible expression system

4. TEMPERATURE-INDUCIBLE Aβ EXPRESSION
SMG-1 is an mRNA surveillance protein.
Temperature inducibility is enabled by a smg-1ts mutation.
mRNA A
3’ UTR
16C
SMG-1 Degradation
20-25C
Translation
A peptide
Degraded RNA

5. HUMAN Aβ IS TOXIC IN WORMS
Aβ1-42 expression in body-wall muscle causes
irreversible paralysis phenotype
0 hr 24 hr

6. MICROARRAY STUDIES Strains: Aβ1-42-expressing animals (CL4176)
GFP::degron-expressing animals (CL2337)
Soluble GFP-expressing animals (CL2179)

7. MICROARRAY STUDIES Experimental Design:
Synchronous animals were grown at 16 ºC for 36 hours.
Transgenes were induced at 25 ºC
Worms were harvested at T0, T4, T8, T12, T16, and T20

8. DIFFERENTIALLY EXPRESSED GENES
IN Aβ ANIMALS

9. DIFFERENTIALLY EXPRESSED GENES
IN Aβ ANIMALS

10. FUNCTIONAL ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES
Aβ-specific genes:
Aging
Insulin signaling
Mitochondrial unfolded protein response
Proteasome degradation pathways
Membrane damage response

11. FUNCTIONAL ANALYSIS OF DIFFERENTIALLY EXPRESSED GENES
Common genes:
Stress response genes
Immune response

12. Aβ TOXICITY AND MEMBRANE DAMAGE
Previous studies suggested Aβ may form membrane pores
Overlap between Aβ and Cry5B genes
Questions:
Intestinal expression of Aβ?
KGB-1 and Aβ toxicity?

13. INDUCTION OF MEMBRANE
DAMAGE RESPONSE

14. kgb-1 RNAi knock down in Aβ animals
A  S tr a in ( C L )
1 0 0
k g b - 1 R N A i C o n t r o l
R N A i
8 0
% N o t p a r a ly z e d
6 0 n1 n2 n3
P value
kgb-1 RNAi 63 51 70
0.0001
Control RNAi 40 53 50
4 0
2 0
1 4
1 6
T im e ( h o u r s )
2 4
2 6

15. kgb-1 RNAi knock down in GFPdeg animals
( C L )
1 0 0
8 0
6 0
4 0
2 0
% N o t p a r a ly z e d n1 n2 n3
P value
kgb-1 RNAi 46 59 67
0.8967
Control RNAi 62 58
102
2 2
2 4
T im e ( h o u r s )
3 2
3 4

16. kgb-1 RNAi knock down in isogenic control animals
C o n tr o l s tr a in ( C L )
1 0 0
8 0
6 0
4 0
2 0
% N o t p a r a ly z e d n1 n2 n3
kgb-1 RNAi 62 44 28
Control RNAi 65 47 41
1 4
1 6
T im e ( h o u r s )
2 4
2 6

17. Aβ1-42 INDUCES aip-1 EXPRESSION
aip-1 expression by Microarrays
aip-1 expression by RT-PCR

18. TRANSCRIPTIONAL REPORTER: aip-1/GFP
Aβ induced
16°C
No Aβ induction

19. KNOCKING DOWN OF aip-1 ENHANCES Aβ TOXICITY
myo-3/Aβ1-42
100 90 80 70 60 50 40 30 20 10 .
% Not paralyzed
26 hrs
28 hrs
aip-1 RNAi
30 hrs
Vector only
32 hrs

20. aip-1 OVER-EXPRESSION DELAYS ALLEVIATES Aβ TOXICITY
myo-3/aip-1 aip-1/aip-1
100 90 80 70 60 50 40 30 20 10
100 90 80 70 60 50 40 30 20 10
% Not Paralyzed
% Not Paralyzed
26 hr
28 hr
30 hr 32 hr
Transgene -
26 hr
28 hr
30 hr 32 hr
Transgene -
Transgene +
Transgene +

21. A COUPLE OF CONTROLS myo-3 /Aβ;aip-1/aip-1 aip-1 RNAi Transgene +
myo-3 /Aβ;aip-1 /GFP
myo-3 /Aβ;aip-1/aip-1 aip-1 RNAi
100 90 80 70 60 50 40 30 20 10
100
% Not Paralyzed
% Not Paralyzed 80 60 40 20
26 hrs
Transgene +
28 hrs 30 hrs
Transgene -
32 hrs
26 hr
28 hr
30 hr 32 hr
Transgene -
Transgene +

22. AIP-1 DECREASES ACCUMULATION OF A

23. A HUMAN HOMOLOGUE IS PROTECTIVE IN WORMS
myo-3/AIRAP myo-3/AIRAPL
100 90 80 70 60 50 40 30 20 10
100 90 80 70 60 50 40 30 20 10 . .
paralyzed
paralyzed
% Not
% Not
24 hrs
26 hrs
29 hrs 31 hrs
Transgene -
Transgene +
Transgene -
Transgene +

24. SUMMARY
Aβ-specific genes include ones involved in aging, insulin signaling, mitochondrial unfolded protein response, membrane damage repair, and proteasome function.
Aβ toxicity appears to be mediated, at least in part, by membrane damage.
AIRAPL, but not AIRAP, is protective against Aβ toxicity.

25. ACKNOWLEDGEMENT
Christopher D. Link, Ph.D. - University of Colorado Boulder
Hassan lab members:
Craig Laufenberg
Aaron Taylor
Brandon Schmidt
NIH and UWM CHS