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

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

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

Control Amyloid Effects of Aβ on erythrocyte morphology

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

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

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

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

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

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.

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

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.

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

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

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

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

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

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

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

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

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

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 :

UNIVERSITY OF CASSINO AND SOUTHERN LAZIO