binding of a beta-amyloid 1-42 peptide to human albumin grifols®
TRANSCRIPT
Poster Presentations P3 P417
suggests that Enox may not act directly on brain Ab in this model. Finally,
since plasma Ab levels remained unchanged, we have no evidence that
Enox acts as a peripheral sink agent. Possibly, the mode of action of Enox
is by improving cerebrovascular perfusion.
P3-250 METALLOTHIONEIN-IIA BLOCKS COPPER
MEDIATED Ab1-40 NEUROTOXICITY
Claire Howells, Adrian K. West, Roger S. Chung, University of Tasmania,
Sandy Bay, Australia. Contact e-mail: [email protected]
Background: The abnormal interaction of copper ions with the amyloid-b pep-
tide (Ab) is associated with aberrant Ab aggregation and reactive oxygen spe-
cies (ROS) production, both of which are toxic to neurons and potentiate the
progression of Alzheimer’s disease (AD). The endogenous astrocyte protein,
metallothionein (MT)-IIA, possesses numerous neuroprotective properties in-
cluding the ability to: sequester metal ions, such as Zn(II) and Cu(I) (Kelly et
al., 1996; Aschner et al., 1997); scavenge free radicals (Sato and Bremner,
1993; Kumari et al., 1998); promote neuroregeneration; and reduce proinflam-
matory responses (Chung et al., 2003). We examined the protective effect of
Zn7MT-IIA against Ab1-40-Cu(II) induced neurotoxicity using an in vitro
model. Methods: Freshly prepared Ab1-40-Cu(II) was applied to cultured rat
cortical neurons, in the presence or absence of different forms of MT. After
24 hours incubation, cellular viability was measured via the alamar Blue� met-
abolic reduction. Neuronal viability was determined as the % survival vs vehi-
cle treated cultures. Results: We determined that Zn7MT-IIA rescued the
neurons from Ab1-40-Cu(II) mediated toxicity (80% vs 30% neuronal via-
bility; p <0.05, ANOVA). The protective effect of MT-IIA was signifi-
cantly higher than Zn7MT-III (80% vs 50% neuronal viability; p < 0.05,
ANOVA). The application of Cu10MT-IIA and carboxyamidomethylated
MT-IIA failed to significantly protect the neurons from Ab1-40-Cu(II) in-
duced neurotoxicity (p>0.05, ANOVA). This indicates that a metal-medi-
ated interaction involving the removal of Cu from Ab is involved in the
protective effect of MT, an idea that has been suggested previously for
Zn7MT-III (Meloni et al., 2008). To investigate this protective mechanism
further, H2O2 was applied directly to cortical neurons (H2O2 is the direct
ROS product of the redox active copper within Ab1-40-Cu(II) (Huang et
al., 1999)). Marked cell death was observed after 30 minute exposure to
100mM H2O2, which could not be blocked by the presence of Zn7MT-
IIA, indicating that MTs protective effect is unlikely to be mediated by di-
rect protection from ROS. Conclusions: These results strongly suggest that
Zn7MT-IIA protects against Ab1-40-Cu(II) induced toxicity by a transmetal-
lation event between Ab1-40-Cu(II) and Zn7MT-IIA. The protection elicited
by Zn7MT-IIA, in addition to its neuroprotective properties, provide great
potential for its therapeutic use in AD.
P3-251 BINDING OF A BETA-AMYLOID 1-42 PEPTIDE TO
HUMAN ALBUMIN GRIFOLS�
Montserrat Costa, Anna Ma Ortiz, Juan Ignacio Jorquera, Instituto Grifols,
S.A., Parets del Valles, Spain. Contact e-mail: [email protected]
Background: Beta-amyloid (Ab) deposits may be the central pathological
process in Alzheimer’s disease. Since there seems to be an equilibrium be-
tween brain and plasma Ab and given that 90% of plasma Ab may be bound
to albumin (1), it might be possible to reduce plasma Ab levels by plasma
exchange with therapeutic albumin, which could in turn translate into brain
amyloid burden reduction (2, 3). The aim of this study is to demonstrate the
capacity of Human Albumin Grifols� to bind a synthetic peptide with the
human amyloid beta peptide 1-42 sequence (sAb1-42). Methods: The capac-
ity of Human Albumin Grifols� to bind human sAb1-42, has been measured
for three different batches, using Surface Plasmon Resonance (SPR). Kinet-
ics and equilibrium features of Human Albumin Grifols� / sAb1-42 peptide
interaction have been analyzed in a Biacore T100 by immobilizing sAb1-42
(Biosource) on a CM5 chip. Known quantities of these samples were ana-
lyzed to obtain kinetic constants for sAb1-42 binding and release. Moreover,
a qualitative comparison of the SPR response has been established by expos-
ing sAb1-42 to plasma samples. Results: Results obtained from three differ-
ent lots of Human Albumin Grifols� show that the association rate constant
(ka) for Human Albumin Grifols�/sAb1-42 is (1.53 6 0.11) x104 M-1s-1,
while the corresponding dissociation rate constant (kd) is (0.026 6 0.004)
s-1. Thus, the equilibrium constant KD (concentration at which 50% of
sAb1-42 is complexed) for Human Albumin Grifols�/sAb1-42 is on average
(1.72 6 0.24) x10-6 M, demonstrating the consistency of this interaction.
Likewise, as expected according to the literature indicating that human am-
yloid beta appears to be able to interact with several plasma proteins, the
SPR response obtained when different plasma samples were analysed was
higher than for isolated albumin. Conclusions: These results indicate that
Human Albumin Grifols� is able to bind to an Ab1-42 peptide with the hu-
man aminoacid sequence. (1): Biere AL et al J Biol Chem, 271: 32916,
1996. (2): Boada M et al Alzheimer’s & Dementia, 4 (Suppl 2): P4-355,
2008. (3): Roca I et al Alzheimer’s & Dementia, 4 (Suppl 2): P4-388,
2008.
P3-252 MICROENCAPSULATED CELLS EXPRESSING
VEGF REDUCE BRAIN AMYLOID LOAD AND
BEHAVIORAL IMPAIRMENT IN ALZHEIMER’S
DISEASE
Carlos Spuch1, Desiree Antequera1, Aitziber Portero2, Gorka Orive3,
Rosa Hernandez3, Jose A. Molina4, Felix Bermejo-Pareja4, Jose L. Pedraz3,
Eva Carro1, 1Research Center. Hospital 12 de Octubre, Madrid, Spain;2Laboratory of Pharmacy and Pharmaceutical Technology, Faculty of
Pharmacy. University of the Basque Country, Vitoria -Gasteiz, Spain;3Neurology service. Hospital 12 de Octubre, Madrid, Spain.
Contact e-mail: [email protected]
Background: Cerebrovascular dysfunction contributes to cognitive decline
and neurodegeneration in Alzheimer’s disease (AD). Vascular endothelial
growth factor (VEGF), an angiogenic protein with important neurotrophic
and neuroprotective actions, is under investigation as a therapeutic agent
for the treatment of neurodegenerative disorders. Cell microencapsulation
technology is a promising strategy for controlled, localised and long term
in vivo delivery of therapeutic peptides to the host. In the present approach,
we have evaluated the use of cell microencapsulation as a tool for the contin-
uous release of VEGF for the treatment of AD. Methods: Assuming that
VEGF plays key roles in brain angiogenesis, neuroprotection and cerebromi-
crovascular exchange of substrates and nutrients, we hypothesized that the
local and continuous release of this angiogenic factor could exert beneficial
effects in the pathogenesis of AD. We used double mutant amyloid precursor
protein/presenilin 1 (APP/Ps1) mice, as a transgenic model of AD, character-
ized by disturbed vessel homeostasis. Results: We report that, 3 months after
stereotaxy implantation of VEGF-microcapsules, brain Ab burden, hyper-
phosphorylated tau and cognitive impairment are attenuated in APP/Ps1
mice. Conclusions: The preliminary data presented herein suggests that mi-
croencapsulated cells secreting VEGF might be an alternative to treat disor-
ders characterized by vascular dysfunction such as AD.
P3-253 ACUTE ADMINISTRATION OF CHF5074, A NOVEL
GAMMA-SECRETASE MODULATOR, IMPROVES
CONTEXTUAL MEMORY IN A TRANSGENIC
MOUSE MODEL OF ALZHEIMER’S DISEASE
Bruno P. Imbimbo1, Luciana Giardino2, Alessandro Giuliani2,
Marco Gusciglio2, Luca Lorenzini2, Gino Villetti1, Laura Calza2, 1Chiesi
Farmaceutici, Parma, Italy; 2BioPharmaNet-DIMORFIPA, University of
Bologna, Ozzano Emilia, Italy. Contact e-mail: [email protected]
Background: CHF5074 is a new gamma-secretase modulator that after
chronic administration has been shown to inhibit brain plaque deposition
and attenuate spatial memory deficit in two different transgenic mouse
models of Alzheimer’s disease (AD) (JPET 2007; 323: 822-30, Br J Pharma-
col 2009 in press). We evaluated the effects of single subcutaneous doses of
CHF5074 on contextual memory in transgenic mice overexpressing the
Swedish mutation of the human amyloid precursor protein. Methods:
Five-month old heterozygous transgenic female mice and aged-matched
transgene-negative littermates (n ¼ 16-27 per genotype per treatment) were
trained and tested on two consecutive days. Freezing behavior was recorded
by a computerized camera. Transgenic mice received two subcutaneous