1. MECHANISMS RESPONSIBLE FOR BETA-AMYLOID-DEPENDENT REDUCTION OF ERYTHROCYTE DEFORMABILITY Francesco Misiti University of Cassino and Southern Lazio Cassino (Italy)

2. Conflict of Interest Francesco Misiti Has no real or apparent conflicts of interest to report

3. o A  are associated with cerebral blood vessels o A  are transferred to blood o A  are produced by platelets o Blood cells are exposed to A  (1-42) and (1-40) o Erythrocytes interact with A  (1-42) o A  alters erythrocyte shape and deformability o A  impairs oxygen delivery to brain Erythrocytes and Alzheimer

4. Control Amyloid Effects of Aβ on erythrocyte morphology

5. Membrane AChE activity as a marker of membrane integrity * P < 0.05 vs. control

6. Objective o Nitric Oxide: a regulatory factor of erythrocyte mechanical properties o Nitric Oxide synthase (eNOS) is expressed in erythrocytes o PKC  and  Caspase 3  responsible for regulation on eNOS function o Pentose Phosphate Pathway (PPP): responsible for erythrocyte antioxidant defense Clarify the role played by Nitric Oxide synthase (eNOS) signaling pathway in the A  -dependent alteration of erythrocyte morphology

7. Western blot analysis showing the conformational species of A  Piacentini et al. J Neurochem (2008 )

8. Western blot analysis showing the conformational species of A  Piacentini et al. J Neurochem (2008)

9. Control Amyloid Immuno-histochemical staining of erythrocyte activated nitric oxide synthase (eNOS) * P< 0.05 vs. control;

10. Time course of the effects of Aβ on nitrite and nitrate levels in erythrocytes suspensions * P < 0.05 vs. control; # P < 0.05 vs. Aβ at 24 hours.

11. Time course of the effects of Aβ on nitrite and nitrate levels in erythrocytes suspensions

12. AChE inhibitors abrogate the effects of Aβ on nitrite and nitrate levels in erythrocytes suspensions * P < 0.05 vs. control; # P < 0.05 vs. Aβ at 24 hours.

13. cytosol membrane Ctr AA + PMA ( 80 kDa ) PKC  + PMA amyloid control PKC  activation by A 

14. cytosol membrane Ctr AA + PMA ( 80 kDa ) PKC  + PMA amyloid control PKC  activation by A 

15. Clementi et al. Int J Biochem Cell Biol. 39: 727-735 (2007) Caspase 3 activation by A  activity

16. Ctr AA Misiti et al. Biochem Cell Biol.86: 1-8 (2008) --32kDa --20 kDa Caspase 3 activation by A  WB

17. Misiti et al. Biochem Cell Biol.86: 1-8 (2008) Mandal et al. JBC 278: 52551-52558 (2003) Band 3 degradation by Caspase 3 Ctr A  A  Ctr AA --32kDa --20 kDa

18. Reduction in antioxidant defense (Pentose Phosphate Pathway) Clementi et al. Int. J. Biochem Cell Biol., 36(10):2066-76 (2004) Ctr A  ** P < 0.05 vs. A  at 24 hours

19. Misiti et al. Biochem Cell Biol.86: 1-8 (2008) A  dependent reduced stimulus for endogenous NO synthesis in the vasculature Ctr A 

20. Conclusions A  /erythrocyte interaction induces : o an imbalance in the antioxidant defense (PPP flux reduction) o Caspase 3 activation o PKC  activation o eNOS down-regulation o NO levels reduction

21. Conclusions A  /erythrocyte interaction induces : o an imbalance in the antioxidant defense (PPP flux reduction) o Caspase 3 activation o PKC  activation o eNOS down-regulation o NO levels reduction

22. Acknowledgements University of Cassino and Southern Lazio- Cassino Cristiana Carelli Alinovi Catholic University-School of Medicine- Rome Bruno Giardina Research National Council (CNR)-ICRM & ISM Beatrice Sampaolese Marco Girasole :

23. UNIVERSITY OF CASSINO AND SOUTHERN LAZIO