Abstracts Punta Cana

ABSTRACTS 13
KEYNOTE LECTURE 13
Peter St. George-Hyslop 13
SESSION I: MODULATORS OF AD NEUROPATHOLOGY 14

CHAIR: FRANK LAFERLA 14
ß-AMYLOID DEGRADATION: THE INSIDE STORY 14
Malcolm A. Leissring 14
CEREBRAL MICROVASCULAR AMYLOID: NEUROINFLAMMATION AND COGNITIVE DEFICITS 14
William E. Van Nostrand 14
REVERSING AGE-DEPENDENT MEMORY IMPAIRMENTS IN A TRANSGENIC MODEL OF ALZHEIMER
DISEASE 15
Frank LaFerla 15
SESSION II: DISEASE MODIFICATION STRATEGIES FOR AD 16

CHAIR: RATAN BHAT 16
GSK3 INHIBITORS FOR ALZHEIMER'S DISEASE 16
Ratan Bhat 16
TRANSLATIONAL BIOMARKERS FOR DISEASE MODIFICATION IN ALZHEIMER'S DISEASE 16
Susanne Fabre 16
PRX-07034 AND PRX-03140 - NOVEL, SELECTIVE AGENTS TARGETING SEROTONIN RECEPTORS FOR
THE TREATMENT OF COGNITIVE DYSFUNCTION 17
Kimberley S. Gannon 17
STABLE TRIMERIC A-BETA PEPTIDES: POTENTIAL VACCINE IMMUNOGENS TO PREVENT ADDL-
TRIGGERED MEMORY DEFICITS 17
Grant Krafft 17
SESSION III: GAMMA-SECRETASE: FUNCTIONALITY AND PHARMACOLOGY 18

CHAIR: ERIC KARRAN 18
-SECRETASE: PAST, PRESENT, FUTURE 18
Alexandra Tolia 18
EXPLOITING THE VARIED PHARMACOLOGY OF THE -SECRETASE COMPLEX 19
Mark Shearman 19
LY450139: TESTING THE AMYLOID CASCADE HYPOTHESIS IN MAN 19
Eric Karran 19
SESSION IV: GENETICS AND TREATMENT STRATEGIES FOR ALZHEIMER'S DISEASE 20

CHAIR: BENGT WINBLAD 20
CLINICAL GENETICS IN ALZHEIMER'S DISEASE 20
Caroline Graff 20
CSF BIOMARKERS IN ALZHEIMER DISEASE CLINICAL TRIALS 20
Khalid Iqbal 20
MEMANTINE LOWERS Β-AMYLOID LEVELS IN NEURONAL CELLS AND TRANSGENIC MOUSE MODELS OF
ALZHEIMER’S DISEASE 21
Pradeep Banerjee 21
TREATMENT STRATEGIES IN SEVERE DEMENTIA 21
Bengt Winblad 21
SESSION V: IMAGING AND MODULATION OF NEUROPATHOLGIES 22

CHAIR: RICHARD COWBURN 22
MECHANISMS OF APOE CONTRIBUTION TO ALZHEIMER'S DISEASE 22
Page 10 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

Angel Cedazo-Minguez 22
A VIEW INTO THE ALZHEIMER'S BRAIN WITH OPTICAL IMAGING 22
Brian J. Bacskai 22
AZAD - A HIGH RESOLUTION PET TRACER FOR IMAGING OF ABETA AMYLOID PLAQUES 23
Samuel Svensson 23
CHOLESTEROL MODIFYING STRATEGIES FOR ALZHEIMER'S DISEASE 23
Richard Cowburn Error! Bookmark not defined.
SESSION VI: IMMUNOTHERAPY FOR ALZHEIMER'S DISEASE 24

CHAIR: DAVE MORGAN 24
COMPLEMENT C3 DEFICIENCY LEADS TO INCREASED PLAQUE BURDEN AND NEURONAL LOSS IN APP
TG MICE BUT DOES NOT INLFUENCE ABETA CLEARANCE BY IMMUNOTHERAPY 24
Cynthia A Lemere Error! Bookmark not defined.
DNA VACCINES FOR ALZHEIMER’S DISEASE WHICH COMBINE NON-SELF T-CELL EPITOPES WITH
MOLECULAR ADJUVANTS THAT UTILIZE THE INNATE IMMUNE SYSTEM TO DRIVE A TH2-MEDIATED
ANTIBODY RESPONSE 25
David H. Cribbs 25
CLEARANCE OF AMYLOID BY ACTIVE IMMUNOTHERAPY HAS DIFFERENT CONSEQUENCES THAN
CLEARANCE BY AN NSAID 26
Dave Morgan 26
SESSION VII: Aß-PEPTIDE: GENERATION, NEUROTOXICITY & TREATMENT STRATEGIES 27

CHAIR: CHRISTIAN HAASS 27
ALZHEIMER’S DISEASE: DEFEATING IT AT THE SYNAPSE 27
Ottavio Arancio 27
IMMUNOTHERAPY AGAINST BETA-AMYLOID IN ALZHEIMER’S DISEASE 28
Roger M. Nitsch 28
A NOVEL HIGH THROUGHPUT SCREEN FOR AΒ POLYMERIZATION INHIBITORS 28
Lars Tjernberg 28
REGULATED INTRAMEMBRANE PROTEOLYSIS IN ALZHEIMER'S DISEASE 29
Christian Haass 29
SESSION VIII: THE ROLE OF BACE INHIBITION IN ALZHEIMER'S DISEASE 30

CHAIR: STEFAN BERG 30
THE Β-SECRETASE BACE1 IN ALZHEIMER’S DISEASE 30
Robert Vassar 30
THE DISCOVERY OF NON TRANSITION-STATE ISOSTERE BACE INHIBITORS 30
Hemaka A. Rajapakse 30
NOVEL GSK3 INHIBITORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES 31
Stefan Berg 31
SESSION IX: GENETIC IMPLICATION OF TAU IN ALZHEIMER'S PATHOGENESIS 32

CHAIR: JOHN HARDY 32
THE ROLE OF PROGRANULIN IN DEMENTIA 32
Rosa Rademakers 32
NOS2 DELETION PROMOTES MULTIPLE PATHOLOGIES IN A NEW MOUSE MODEL OF ALZHEIMER’S
DISEASE 32
Michael P. Vitek 32
GENETIC DISSECTION OF THE ETIOLOGIES AND PATHOGENESES OF ALZHEIMER’S AND PARKINSON’S
DISEASES AND RELATED DISORDERS 33
John Hardy 33
SESSION X: DEGRADATION, AGGREGATION AND PHOSPHORYLATION: PATHOGENIC POTENTIAL AND

THERAPEUTIC OPPORTUNITIES IN ALZHEIMER'S DISEASE 34
Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 11

CHAIR: KAREN DUFF 34
ROLES OF HEAT SHOCK PROTEIN 90 IN MAINTAINING AND FACILITATING THE DEGENERATIVE
PHENOTYPE IN TAUOPATHY 34
Gabriela Chiosis 34
MECHANISMS OF TAU AGGREGATION: HOW DO POST-TRANSLATIONAL MODIFICATIONS AND TAU
SPLICING MUTATIONS PROMOTE NEUROFIBRILLARY LESION FORMATION? 35
Jeff Kuret 35
ABERRANT PHOSPHORYLATION AS A PATHOGENIC MECHANISM IN ALZHEIMER'S DISEASE 35
Karen Duff 35
POSTERS 36
LIFELONG CALCIUM DISRUPTIONS IN ALZHEIMER’S DISEASE MICE: NORMAL AGING VS. PATHOLOGICAL
SIGNALING CHANGES IN NEURONS 36
Grace E. Stutzmann 36
UNFOLDED P53 PROTEIN AS A PERIPHERAL AD MARKER 36
Daniela Uberti Error! Bookmark not defined.
CHARACTERISATION OF CELL-PERMEABLE BACE-1 INHIBITORS IN PRIMARY NEURONS 37
Christiane Volbracht 37
PREVENTION OF FIBRIL FORMATION AND ABETA-MEDIATED NEUROTOXICITY BY ANTI-D-PEPTIDE
ANTIBODIES 38
M. Yu 38
WHERE EXACTLY IN THE CELL DO THE SECRETASES CLEAVE? TARGETING THE LOCATION FOR THERAPY
38
Lawrence Rajendran 38

ABSTRACTS
KEYNOTE LECTURE
KEYNOTE LECTURE
Peter St. George-Hyslop
University of Toronto
Genetic epidemiological studies have suggested that approximately 40% of the variance in the
risk for Alzheimer Disease (AD) can be attributed to inherited factors. These studies have also
indicated that amongst these genetic risk factors, only a minority are inherited as simple
autosomal dominant traits. The majority of risk factors are inherited in a more complex fashion in
which there are likely to be combinations of several different genetic factors, each with a low
effect size. Furthermore, different allelic variants of a given genetic factor are also likely to exist
(again, with varying but low effect sizes). Recent molecular genetic studies have identified at
least five different genes associated with risk for AD (PS1, PS2, APP, SORL1 and APOE). These
genes probably account for about half of the genetic factors, with APOE accounting for the
majority of this effect. It is presently unclear how many remaining AD-susceptibility genes exist. It
is likely that there will still be a few families with very rare mutations in single genes (but these will
likely be much less frequent than APP, PS1 or PS1). The remaining genes are likely to be
incompletely penetrant, with small effect sizes (probably weaker than that of APOE). Current
estimates have suggested that there may be as many as 7-8 such additional genes. However, if
the effect sizes of these genes are smaller than APOE, this number may represent an
underestimate.
The biochemical and molecular biological analyses of the known AD-causing genes currently imply
that the common biochemical property shared by these genes is that they impact the processing
of the amyloid precursor protein. If true, this hypothesis would suggest two testable questions.
First, if the disease-causing genes all act within the same biochemical pathway, they should have
demonstrable interactions. Second, blocking the production and/or aggregation of amyloid should
prevent, retard, or even reverse features of the disease.
Recent experimental evidence supports positive answers to both of these questions. Clear
evidence of gene interactions can now be found in cell-based, animal model and human clinical
studies. Similarly, several pre-clinical studies in animal models suggest that blocking the
formation and/or aggregation and/or accelerating the removal of A may be effective. The results
of ongoing clinical trials in humans will obviously be needed to fully resolve this question, but
emerging data seems promising. Nevertheless there are still several unaddressed questions
including: 1) how the chronic accumulation of A leads to the accumulation of tau deposits inside
neurons; 2) how the combined effects of A and tau aggregates cause neuronal dysfunction and
death; and 3) whether treatments directed solely at Aβ will be sufficient to stop AD once
established.

Session I: Modulators of AD Neuropathology
Chair: Frank LaFerla
ß-AMYLOID DEGRADATION: THE INSIDE STORY
Malcolm A. Leissring*, Ji Zhao, Lilin Li, Qun Lu & Malwina Huzarska

The Scripps Research Institute
Proteases that degrade the amyloid-ß protein (Aß), which accumulates abnormally in Alzheimer’s
disease (AD), have emerged as potent regulators of cerebral Aß levels and amyloidogenesis in
vivo. However, whereas most Aß-degrading proteases are present in the extracellular space,
many are localized to diverse intracellular compartments. In animal models that develop both Aß
plaques and neurofibrillary tangles, intracellular accumulation of Aß has been identified as a key
antecedent to tangle formation. Together, these observations suggest that different Aß-degrading
SESSION I: Modulators of AD Neuropathology
proteases may contribute differentially to different aspects of AD pathogenesis by virtue of

regulating distinct intracellular pools of Aß. To address this topic, we studied two Aß-degrading
proteases with unique intracellular localization profiles: cathepsin D (CatD), an aspartyl protease
localized principally within lysosomes; and insulin-degrading enzyme (IDE), a zinc-metalloprotease
localized to cytosol and mitochondria, in addition to the extracellular space. Here we report that
CatD knockout mice exhibit pronounced increases in endogenous Aß42 levels(~140%, the largest
detected in any protease knockout mouse line), but—remarkably—show no changes in Aß40.
These results suggest that Aß42 is selectively degraded within lysosomal compartments in vivo,
and support the hypothesis that qualitatively different consequences can arise from defects in
individual Aß-degrading proteases. We also investigated the relative roles of extracellular vs.
intracellular pools of IDE with the aid of potent IDE inhibitors developed within our laboratory.
Relative to cell-impermeant IDE inhibitors, structurally similar cell-permeant IDE inhibitors
produced substantially larger increases in the levels of Aß secreted naturally from cells stably
overexpressing the ß-amyloid precursor protein, suggesting that intracellular pools of IDE
contribute in important ways to net Aß levels via degradation within unidentified subcellular
compartments. Moreover, we report that IDE-KO mice show profound disturbances in insulin-
signaling pathways that are predicted to contribute to tangle formation in vivo. Together, our
results support the hypothesis that defects in distinct Aß-degrading proteases, by virtue of
differences in their subcellular localization and/or functional roles, may contribute differentially to
different aspects of AD pathogenesis in vivo.
CEREBRAL MICROVASCULAR AMYLOID: NEUROINFLAMMATION AND COGNITIVE DEFICITS
William E. Van Nostrand, Rong Fan, Feng Xu, Mary Lou Previti, Alicia M. Grande, John K.
Robinson and Judianne Davis
Stony Brook University
Cerebral microvascular amyloid ß protein (Aß) deposition and associated neuroinflammation is

increasingly recognized as an important component leading to cognitive impairment in
Alzheimer’s disease and related cerebral amyloid angiopathy (CAA) disorders. Transgenic mice
expressing the vasculotropic Dutch/Iowa (E693Q/D694N) mutant human Aß precursor protein in
brain (Tg-SwDI) accumulate abundant cerebral microvascular fibrillar amyloid deposits and exhibit
robust neuroinflammation. We sought to determine if the unique amyloid pathology of Tg-SwDI
mice was associated with deficits in behavioral performance. Our results indicate that Tg-SwDI
mice were impaired in the performance of the Barnes maze learning and memory task at 3 and

12 months of age. While more widespread cerebral microvascular Aß pathology was evident in
SESSION I: Modulators of AD Neuropathology

older animals, the evaluation of the Aß pathology in the 3 months old transgenic animals revealed
specific accumulation of microvascular amyloid and markedly elevated numbers of reactive
astrocytes and activated microglia in the subiculum region.
Next, we investigated the effect of the anti-inflammatory drug minocycline on Aß accumulation,

neuroinflammation, and behavioral deficits in Tg-SwDI mice. Twelve months old mice were treated
with saline or minocycline by intraperitoneal injection every other day for a total of four weeks.
During the final week of treatment the mice were tested for impaired learning and memory.
Brains were then harvested for biochemical and immunohistochemical analysis. Minocycline
treatment did not alter the cerebral deposition of Aß or the restriction of fibrillar amyloid to the
cerebral microvasculature. Similarly, minocycline-treated Tg-SwDI mice exhibited no change in
the levels of total Aß, the ratios of Aß40 and Aß42, nor in the amounts of soluble, insoluble, or
oligomeric Aß compared to the saline-treated control Tg-SwDI mice. In contrast, the numbers of
activated microglia and levels of interleukin-6 were significantly reduced in minocycline-treated
Tg-SwDI mice compared to saline-treated Tg-SwDI mice. In addition, there was a significant
improvement in behavioral performance of the minocycline-treated Tg-SwDI mice. These finding
suggest that anti-inflammatory treatment targeted for cerebral microvascular amyloid induced
microglial activation can improve cognitive deficits without altering the accumulation and
distribution of Aß.
REVERSING AGE-DEPENDENT MEMORY IMPAIRMENTS IN A TRANSGENIC MODEL OF

ALZHEIMER DISEASE
Frank LaFerla
University of California, Irvine
Progressive memory loss and cognitive dysfunction are the obligate clinical features of Alzheimer
disease. Identifying their molecular determinants presently requires the use of animal models,
such as the 3xTg-AD mice, which develop both amyloid and tangle pathology. We used several
behavioral paradigms to characterize their cognitive phenotype. The experimental design
included both longitudinal and cross-sectional analysis, enabling us to determine the influence of
repeated training on subsequent cognitive performance. We first show that pre-pathologic mice
are unimpaired. The earliest impairment manifests at 4 months and correlates with the
accumulation of intraneuronal Aß, with the notable absence of plaques or tangles. Clearance of
the intraneuronal Aß by immunotherapy rescues the early cognitive deficits. Moreover, we
explored the contribution of early and repeated learning in these mice. The mice were trained in a
longitudinal design prior to the emergence of Aβ and tau pathology. Their performance was
compared to mice trained in a cross-sectional design and mice trained longitudinally after the
emergence of pathology. Thus, we were able to directly explore the reciprocal relationship
between learning and memory and Aß and tau pathology. We find that 3xTg-AD mice trained
longitudinally show a significant acquisition and retention benefit at 9 and 12 months of age.
This training effect was dependent on learning, rather than simple exposure to a novel testing
environment without a learning component. In contrast, naïve 3xTg-AD mice showed a
progressive, step-wise deterioration in cognition, in line with advancing neuropathology.
Longitudinally-tested mice contained lower levels of Aβ42 and somatodendritic tau after testing.
These findings provide a thorough analysis of alterations in cognition in relation to emerging
neuropathology, and show that repeated, acute learning paradigms can significantly benefit
future cognition and decrease pathology in mouse models of AD.

Session II: Disease Modification Strategies for AD
Chair: Ratan Bhat
GSK3 INHIBITORS FOR ALZHEIMER'S DISEASE

Ratan Bhat and Stefan Berg
AstraZeneca R&D Södertälje
The development of dementia highly correlates with the formation of intracellular neurofibrillary
tangles (NFT), and brain atrophy. Abnormal hyperphosphorylation of the microtubule associated
protein tau results in cytoskeletal abnormalities and its subsequent post-translational
SESSION II Disease Modification Strategies for AD
modification result in the formation of paired helical filament (PHF), the major constituents of the
NFT. GSK3 a serine / threonine kinase has been implicated as a major kinase in the aberrant
hyperphosphorylation of tau leading to NFTs in AD.
GSK3 phosphorylates the majority of PHF phosphorylation sites in AD brain. Transgenic mice
overexpressing GSK3β exhibit extensive pre-tangle pathology such as tau hyperphosphorylation at
PHF sites, neurodegeneration, gliosis and cognitive deficits, thereby providing a strong link
between increased GSK3 activity and tau pathology in AD. Accordingly, inhibition of pre-tangle
pathology via GSK3 inhibition would be expected to slow down the progression of NFT formation
and neurodegeneration in AD.
We report here the discovery of a series of GSK3 inhibitors. Several compounds were found to be
selective and potent inhibitors of GSK3 in vitro and in vivo. We provide evidence for cellular and in
vivo reduction of tau phosphorylation and microtubule deficits in brains of mice overexpressing
the tau mutation P301L. In vivo exposure studies showed that these compounds are brain
permeable. The pyrazines may thus have important implications in the treatment of AD and
related dementias.
TRANSLATIONAL BIOMARKERS FOR DISEASE MODIFICATION IN ALZHEIMER'S DISEASE

Susanne Fabre
The role of biomarkers as success factors in drug discovery is becoming more evident. An
important task is to identify and characterize drug target-linked biochemical end-points in animal
and human peripheral compartments such as plasma and CSF. These biochemical endpoints
serve to confirm that the drug affects the target (PoM) and to estimate effect on pathogenic
processes (PoP). Validated endpoints can be used further for establishment of PK/PD relationship
and dose selection in clinical studies. Furthermore, biochemical biomarkers may be important to
use in stratification for homogeneous patient groups, for example in clinical trials using MCI
patients with a high probability to convert to AD.
In AD, CSF sampling has been used for diagnostic purposes and exploratory research for decades.
The combination of T-Tau, P-Tau and Aβ40 and 42 has been evaluated as diagnostic biomarkers
for early disease. If validated further, these biochemical biomarkers will be useful for
distinguishing MCI patients who will progress to AD. Furthermore, monitoring Aβ fragments in CSF
and plasma looks promising as proof of mechanism markers for treatment targeting amyloid
processing and/or clearance. However, whether tau and p-tau are suitable for monitoring disease
progression during treatment remains to be shown.
Neurofilaments and Isoprostanes are examples of other markers present in CSF, that may reflect
neuronal damage and therefore be useful to study positive effects on neurodegeneration in
general. Understanding of how these markers are affected by treatment needs to be explored

both in preclinical as well as clinical settings. Other biomarkers suitable for translation in AD is
SESSION II: Disease Modification Strategies for AD

imaging techniques such as volumetric MRI or PET.
PRX-07034 and PRX-03140 - NOVEL, SELECTIVE AGENTS TARGETING SEROTONIN RECEPTORS

FOR THE TREATMENT OF COGNITIVE DYSFUNCTION
Kimberley S. Gannon, Ganesh Iyer, Ed Parsons, Sharon Shacham
EPIX Pharmaceuticals, Inc.
EPIX Pharmaceuticals has two novel, highly selective serotonin receptor-targeted drugs in clinical
development for the treatment of Alzheimer’s disease and other cognitive disorders. PRX-07034
is a potent (Ki = 4nM) serotonin subtype 6 (5-HT6) receptor antagonist in early clinical trials.
Preclinical studies have demonstrated that PRX-07034 significantly improved cognitive
functioning in a variety of learning and memory models, including scopolamine-induced memory
deficits, age-induced memory deficits, working memory and executive function/cognitive flexibility.
PRX-07034 also enhanced the efficacy of donepezil (Aricept®) in a preclinical test of recognition
memory. 5-HT6 receptor antagonists have been shown to modulate several neurotransmitter
systems, including acetylcholine, involved in learning and memory. A 28-day Phase 1 multiple
ascending dose clinical trial of PRX-07034 was recently completed. The study was designed to
assess the safety and tolerability of PRX-07034 in obese, otherwise healthy volunteers, as 5-HT6
antagonism has also been proposed as a treatment for obesity. Cognitive functioning was
assessed as a secondary endpoint using the CogScreenTM test battery. Initial, top-level results of
the study will be presented.
A second drug candidate, PRX-03140, is a selective 5-HT4 partial agonist (Ki = 30nM) that is
currently undergoing clinical testing in Alzheimer’s disease patients. Activation of 5-HT4 receptors
has been shown to increase the release of acetylcholine in the brain (potentially synergistic with
cholinesterase inhibitors), as well as to stimulate secretion of the non-amyloidogenic soluble form
of the amyloid precursor protein, implicating a dual cholinergic/disease-modifying mechanism for
the treatment of Alzheimer’s disease and other dementias. Clinical data from three Phase 1 trials
have indicated that PRX-03140 is well tolerated and has produced alterations in brain wave
activity (alpha:theta ratio by qEEG) in patients with mild-to-moderate Alzheimer's disease that are
consistent with changes seen with cholinesterase inhibitors. A Phase 2a study of PRX-03140
alone and in combination with donepezil (Aricept®) is currently underway, with data expected at
the end of 2007.
STABLE TRIMERIC A-BETA PEPTIDES: POTENTIAL VACCINE IMMUNOGENS TO PREVENT ADDL-

TRIGGERED MEMORY DEFICITS
Grant Krafft
Acumen Pharmaceuticals
Background: The picture of Alzheimer’s disease has changed dramatically over the past decade,
as emerging data now implicate amyloid β-derived diffusible ligands (ADDLs) as the key memory-
compromising molecules responsible for AD. We now understand that ADDLs assemble from
elevated Aβ42, and they selectively attack dendritic spine receptors on a subset of neurons. The
ensuing signals disrupt long-term potentiation (LTP), leading to compromised learning and
memory. In connection with our collaborative ADDL-directed therapeutic antibody program, we
developed novel trimeric Aβ peptide immunogens capable of generating antibodies with potent
ADDL assembly blocking activity. Objective(s): This study targets the design and pre-clinical
development of trimeric vaccine immunogens that trigger production of selective ADDL assembly-

blocking antibodies, without accompanying anti-monomer or anti-fibril antibodies. Methods: We
have deployed molecular dynamics and 2D NMR studies to reveal key structural aspects of stable
Aβ analog trimers, allowing design of vaccine immunogens with optimized properties. Results:
SESSION III: Gamma-secretase: Functionality and Pharmacology
Several novel trimeric immunogens are capable of triggering polyclonal antibodies with potent
ADDL assembly blocking activity and minimal anti-monomer activity. Studies are continuing to
generate an optimized IND-candidate immunogen. Conclusions: We have demonstrated the
fesibility of generating vaccine immunogens that generate a trimer-specific, ADDL assembly
blocking immune response. We believe that vaccines targeting Aβ trimers will be highly effective
AD therapeutics with great potential to prevent AD.
Session III: Gamma-secretase: Functionality and

Pharmacology
Chair: Eric Karran
The Amyloid Cascade Hypothesis posits that the production of the Abeta peptide is a central event
in the Alzheimer’s disease (AD) process. The gamma secretase complex is an unusual enzyme
that releases the Abeta peptide from the C-terminal stump of the amyloid precursor protein
following BACE cleavage. The gamma secretase cleavage site requires intra-membranous
proteolysis.
Work on inhibitors of gamma secretase, as potential therapeutic agents for AD, started well
before the protein components of the complex were identified. In the last few years, the nature of
the enzyme, its mechanism of action, its sub-unit organization and the effects of different types of
low molecular weight inhibitor molecules has started to be elucidated. In parallel, gamma
secretase inhibitors are entering advanced clinical testing in AD patients. This session will review
our knowledge of the gamma secretase complex from basic biochemistry, through pharmacology
and finally to preclinical and clinical testing.
-SECRETASE: PAST, PRESENT, FUTURE
Alexandra Tolia and Bart De Strooper

K.U.Leuven and VIB
Since its initial discovery more than a decade ago, the -secretase complex has been one of the
most attractive but challenging drug targets for Alzheimer’s Disease. This protease provides the
final cut in the generation of the Aβ peptide by cleaving within the transmembrane domain of APP.
The catalytic core of the complex resides on an aspartic protease, Presenilin, while three more
membrane proteins, namely Nicastrin, APH-1 and PEN-2, are essential and sufficient for its
activity. We will review how at least 4 different complexes contribute to the proteolytic function. -
Secretase is a particularly challenging drug target because it cleaves additional proteins, most
importantly Notch, which is crucial in essential physiological processes in development but also in
adult tissues. On the other hand, recent advances in the field of Regulated Intramembrane
Proteolysis, in combination with novel biochemical approaches like cysteine scanning
mutagenesis, have provided more insight into the structure and catalytic mechanism underlying
the activity of the -secretase complex, setting off the beginning of a new era in the design of
specific inhibitors.

EXPLOITING THE VARIED PHARMACOLOGY OF THE -SECRETASE COMPLEX
SESSION III: Gamma-secretase: Functionality and Pharmacology

Mark Shearman
Merck
Recent advances in identifying the components and structure of the -secretase complex have
helped define this enzyme as a therapeutic target for Alzheimer's disease. Amongst the recent
progress that has been made is the definition of active-site directed prototypical inhibitors, which
appear to block the processing of multiple substrates, as well as inhibitors which bind at an
alternative site on the enzyme. Both classes of molecule are distinct from -secretase
modulators, which alter the site of substrate cleavage rather than inhibiting the enzyme. Such
differences in the mode of action have been explored with conventional pharmacological tools as
well as by mutagenesis, and are faithfully recapitulated in vitro and in vivo. Studies aimed at
deciphering the mechanistic details of the complex will be described, as well as approaches to
identifying compounds with favorable activity profiles, and their exploitation to develop important
new Aβ peptide lowering classes of therapeutics for Alzheimer's disease.
LY450139: TESTING THE AMYLOID CASCADE HYPOTHESIS IN MAN
Eric Karran
Eli Lilly & Company
Well before the nature of the γ-secretase complex was described, cell-based assays were used to
identify compounds that inhibited the production of Abeta peptide. Using these assays to drive
SAR led to the development of a functional γ -secretase inhibitor, LY450139. LY450139 has
been demonstrated to reduce Abeta in a variety of pre-clinical animal models including transgenic
APPV717F mice, non-transgenic mice and beagle dog.
Using inhibition of plasma Abeta as a biomarker to bridge from preclinical studies, LY450139 has
completed Phase 2 testing and is poised to enter Phase 3 trials for Alzheimer’s disease. This talk
will review the progression of this compound from cell-based assay systems, to preclinical studies
and finally to testing in humans.

Session IV: Genetics and Treatment Strategies for
Alzheimer's Disease
Chair: Bengt Winblad
CLINICAL GENETICS IN ALZHEIMER'S DISEASE
Caroline Graff
SESSION IV: Genetics and Treatment Strategies for AD
Karolinska Institute
The familial forms of Alzheimer’s disease (FAD) are in principle indistinguishable from the
common sporadic forms of AD with the exception for the familial clustering and the age at onset.
Thus, the rare familial forms may be used as models for the sporadic forms and help us
understand the pathogenic mechanisms of neurodegeneration. Three genes have been identified
which cause autosomal dominant AD and they are all functional parts of the amyloidogenic
pathway i.e. amyloid precursor protein (APP), presenilin-1 (PSEN1) and presenilin-2 (PSEN2).
However, the majority of autosomal dominant AD families have mutations in as yet unidentified
genes and we still do not know if these unknown genes are involved in the same pathway(s).
Much of our understanding of the molecular mechanisms of AD has used the familial mutations
to model the disease including mouse models and in vitro studies. Thus, finding causal mutations
in new genes would be highly valuable. We have performed a whole genome linkage analysis in
Swedish AD families in an effort to identify novel AD genes. One of the outcomes of this study is
that the phenotype within families may vary both with respect to the clinical phenotype and also
with respect to the morphological characteristics at autopsy. This clearly illustrates the complexity
of the disease and the need for better diagnostic tools.
CSF BIOMARKERS IN ALZHEIMER DISEASE CLINICAL TRIALS
Khalid Iqbal
New York State Institute for Basic Research In Developmental Disabilities
Alzheimer disease (AD) is multifactorial, heterogeneous and involves different disease

mechanisms. During the last twenty years, sufficient knowledge has been gained on the
molecular mechanisms of the two hallmark lesions, the β-amyloidosis and the neurofibrillary
degeneration, of AD, and currently and appropriately, the generation of a number of
neuroprotective therapeutic drugs are underway by the pharmaceutical industry. However, the
successful development of these therapeutic drugs will depend on ways to stratify test subjects
and the availability of outcome measures with which the neurodegeneration involved can be
evaluated in life. Towards these aims, we have identified five different subgroups of AD based on
the CSF levels of Aβ 1–42, total tau and ubiquitin, the three major proteins associated with the
histopathological hallmarks of the disease. Each AD subgroup correlates with a different set of
specific clinical symptoms. Stratification of AD patients into the five identified subgroups by CSF
molecular markers can help evaluate the specificity and efficacy of therapeutic drugs to both
different disease subgroups and to the total AD group, thereby substantially enhancing the
success rate of clinical trials. Furthermore, the CSF levels of molecular markers may serve as
useful outcome measures to demonstrate the efficacy of test drugs towards inhibiting the AD
neurodegeneration. (Supported in part by the New York State Office of Mental Retardation and
Developmental Disabilities, NIH grants AG019158, AG028538 and a grant from Alzheimer’s
Association, Chicago, IL.)

MEMANTINE LOWERS Β-AMYLOID LEVELS IN NEURONAL CELLS AND TRANSGENIC MOUSE
SESSION IV: Genetics and Treatment Strategies for AD

MODELS OF ALZHEIMER’S DISEASE
Pradeep Banerjee1, Debomoy Lahiri2, David Holtzman3, Thomas Wisniewski4, Sandeep Gupta1,
Frank LaFerla5
1ForestResearch Institute, Jersey City, NJ; 2Department of Psychiatry, Indiana University School of
Medicine, Indianapolis, IN;3Department of Neurology, Washington University, St. Louis; 4New York University
School of Medicine, New York, NY; 5University of California, Irvine, CA
Memantine, an uncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, is approved

for the treatment of moderate to severe Alzheimer’s disease (AD). One predominant
neuropathological feature of AD is the extracellular deposition of neurotoxic beta-amyloid (Aβ)
plaques. We have reported earlier that memantine rescues brain cells from the neurotoxic effects
of Aβ. Here we show that memantine - at therapeutic doses -lowers the levels of Aβ peptides in
neuronal cells and in transgenic-AD mice with high brain levels of Aβ. We also found that
memantine reduces the cortical and hippocampal plaque burden in APP/PS1 mice and improves
cognitive performance of double (APP/PS1) and triple transgenic (APP/PS1/tau) mice in several
behavioral tests. These data provide strong preclinical evidence for disease-modifying effects of
memantine in AD.
TREATMENT STRATEGIES IN SEVERE DEMENTIA
Bengt Winblad
Karolinska Institutet
In developed countries, where elderly patients constitute an escalating proportion of the

population, the prevalence of AD is increasing. Treatment regimens throughout the different
stages of dementia vary, with objectives broadening as the disease progresses and patients
experience a deterioration of their symptoms. In the early stages of AD, an active patient role is
encouraged and residual abilities are important. In severe dementia, in addition to treating the
patient, means of reducing the burden on both the caregiver and health system must be
considered.
The pharmacoeconomic aspects of dementia are important. The cost of treating AD is high and
the financial burden increases as the disease progresses. Hospitalisation and nursing home
placement account for the most significant direct costs; therapies that delay the
institutionalisation of patients will result in significant cost savings.
Acetylcholinesterase inhibitors have been approved for the treatment of mild-to-moderate AD. Two
studies have been positive in moderately severe and severe AD. The recent study in Swedish
nursing home residents demonstrates the clinical utility of donepezil in a very severe population
(patients with Mini-Mental State Examination scores of 1 to 10 inclusive). Significant differences
favouring donepezil treatment were observed at the end point of this 6-month double-blind,
placebo-controlled study on the 2 primary outcome measures; the Severe Impairment Battery
(SIB) and modified Alzheimer’s Disease Cooperative Study Activities of Daily Living Inventory for
Severe AD (ADCS-ADL-severe).
The NMDA-antagonist memantine has shown to be effective in moderately severe-to-severe AD. A
pharmacoeconomic study showed that treatment with memantine reduced caregiver time and
delayed institutionalisation, i.e. was cost effective. Furthermore a study in moderately severe and
severe AD with the combination donepezil and memantine was very positive. Memantine is the
only drug approved on the indication severe Alzheimer’s disease by European authorities, while
both memantine and donepezil are approved on the same indication by US authorities.

Session V: Imaging and Modulation of Neuropatholgies
Chair: Richard Cowburn
MECHANISMS OF APOE CONTRIBUTION TO ALZHEIMER'S DISEASE

SESSION V: Imaging and Modulation of Neuropathologies
Angel Cedazo-Minguez
Apolipoprotein E (apoE) is a key molecule in lipid metabolism that has important roles in
neurobiology. From the three human isoforms of apoE (E2, E3 and E4), apoE4 is the major known
risk factor for Alzheimer’s disease (AD), and has been associated with several other
neurodegenerative disorders. Although the mechanisms underlying the actions of apoE4 in
neurodegeneration are poorly understood, increasing evidences strongly suggest that apoE4 has
direct detrimental effects, including direct toxicity, on neurons. In the present work, we show that
apoE4 is toxic by a mechanism mediated by the activation of CamKII and Ask1 and by the
translocation of Daxx, the death-associated protein, to the cytosol. This toxicity was prevented by
overexpression of thioredoxin-1 or glutaredoxin-1, suggesting that it can be reversed by Ask1
inhibition. Furthermore, overexpression of DJ-1, the redox-sensitive protein that binds Daxx in the
nucleus, also protected against apoE4 toxicity. Interestingly, mutations in DJ-1 cause familial
forms of Parkinson’s Disease (PD), and some reports have also associated PD and the presence
of apoE4. In this work, we describe a novel mechanism by which apoE4 could contribute to the
pathogenesis of neurodegenerative disorders.
A VIEW INTO THE ALZHEIMER'S BRAIN WITH OPTICAL IMAGING
Brian J. Bacskai
MassGeneral Institute for Neurodegenerative Diseases
Alzheimer’s disease is an illness that can only be diagnosed with certainty following post-mortem
examination of brain tissue. Amyloid-beta plaques are one of the pathological hallmarks of the
disease. Therefore, an imaging technique that allowed direct examination of this CNS pathology
would allow early diagnosis of the disease, as well as provide a quantitative endpoint for
evaluating experimental therapeutics. Using multiphoton microscopy in transgenic mouse
models of Alzheimer’s disease, we have imaged amyloid-beta plaques longitudinally in living mice
to characterize the natural history of single, identified senile plaques. We have also evaluated
several therapeutic strategies aimed at modifying this pathology. Efforts towards a non-invasive
optical imaging approach using near infrared fluorescence and diffuse optical tomography in
mouse models are underway. We have generated a number of fluorescent contrast agents and
developed novel instrumentation for quantitative imaging of amyloid pathology in the brain. All
together, multiphoton microscopy has provided a window to the Alzheimer’s brain, and will
continue to advance efforts towards clinically relevant diagnostic imaging.

AZAD - A HIGH RESOLUTION PET TRACER FOR IMAGING OF ABETA AMYLOID PLAQUES
SESSION V: Imaging and Modulation of Neuropathologies

Samuel Svensson
The presence of β-amyloid plaques is the hallmark of Alzheimer disease and the marker by which
diagnosis is confirmed posthumously. PET radioligands that bind selectively to amyloid offer a
unique tool for allowing the assessment of both the severity of disease in vivo and the
effectiveness of new drugs that aim to reduce plaque load.
AZAD has been identified using radioligand binding screening as a selective amyloid binding
ligand with high affinity. The preclinical profile of AZAD suggests its suitability as a PET radioligand
for amyloid plaque detection in man. Compared to previously published PET radioligands it shows
significantly lower nonspecific binding and improved binding kinetics with a clear reversible
binding profile. The low nonspecific binding, in particular in brain white matter, shown in vitro is
translated to both transgenic mice and in monkey. We anticipate that [11C] labeled AZAD will show
an improved PET signal in AD brains.
CHOLESTEROL MODIFYING STRATEGIES FOR ALZHEIMER'S DISEASE
Johanna Lindquist, Susanne Gustavsson, Johanna Sjödin, Jan Neelissen, Lars I Andersson
Susanne Fabre, Anders Bäckström, Britt-Marie Swahn, Gvido Cebers, Richard Cowburn
Much recent evidence suggests a link between cholesterol and Alzheimer's Disease (AD).
Epidemiological studies have shown that elevated mid-life cholesterol confers an increased risk of
developing AD, whereas inhibitors of cholesterol biosynthesis, statins, reduce the prevalence of
AD up to 70%. Mechanistic studies in cell culture, rabbits and transgenic mice indicate that the
production of Abeta is sensitive to cellular cholesterol. The precise mechanism whereby
cholesterol affects Abeta generation and transport has not been fully elucidated.
Nuclear receptors Liver X Receptors (LXRalpha and LXRbeta) are implicated in regulating central
and peripheral cholesterol homeostasis. LXRbeta is the predominantly expressed form in brain,
both in neurons and astrocytes. Cholestrol can be converted to oxysterols by mitochondrial
hydroxylases, Cyp46 and Cyp27. Oxysterols act as ligands for LXR which in turn regulates the
expression of a number of genes involved in lowering cholesterol levels.
Recent in vivo studies suggest that LXR agonists can reduce Abeta levels in the brains of APP
transgenic mice. We tested this hypothesis by profiling two chemically different LXR agonists
(T0901317 and GW3965) both in vitro and in vivo for Abeta lowering effects. In vitro profiling
showed that neither agonist lowered Abeta release from APPSwe HEK-293 cells. T0901317 has
been recently suggested to modulate gamma-secretase processing of APP. Using a cell
membrane assay we found no activity of T0901317 against gamma-secretase. In vivo testing of
both T0901317 and GW3965 in acute and long-term paradigms in APP Tg2576 mice and Guinea
pigs failed to show reductions in plasma soluble Abeta or in brain soluble and insoluble Abeta.
Both agonists gave excellent brain exposure and hit the target as shown by strong induction of
ABCA1, ABCG1 and ApoE expression.
We conclude that although LXR is a pharmacologically and mechanistically attractive target, LXR
agonists do not have marked effects on Abeta levels and lack potential as Alzheimer’s disease
modifiers.

Session VI: Immunotherapy for Alzheimer's Disease
Chair: Dave Morgan
It is likely that the first test of the amyloid hypothesis of Alzheimer's disease in human patient
populations will involve immunotherapy. Active and passive immunization are remarkably
effective in reversing the phenotype in many mouse models of amyloid deposition. Yet active
vaccination trials in patients resulted in a subset of patients having adverse reactions. Still,
multiple groups are continuing to pursue this strategy either with passive immunization with
monoclonal antibodies, or what hopefully will be safer active vaccines. This session will update
attendees on recent results in mouse models of amyloid deposition with respect to safer vaccine
SESSION VI: Immunotherapy for Alzheimer's Disease
preparations and mechanisms of antibody mediated amyloid clearance. Development of

sophisticated vaccination strategies that use Aß B cell epitopes but alternative T cell epitopes will
be presented. Data arguing against a role of complement activation and/or inflammation as
contributing to antibody mediated amyloid clearance will be presented. Finally the potential
problem of vascular amyloid accumulation and vascular leakage with either active or passive
immunization will be discussed. All of these data raise important questions regarding the
mechanisms by which immunotherapy prevents/clears amyloid deposits in brain, and what steps
might be taken to maximize patient safety in clinical trials of immunotherapy.
COMPLEMENT C3 DEFICIENCY LEADS TO INCREASED PLAQUE BURDEN AND NEURONAL LOSS

IN APP TG MICE BUT DOES NOT INLFUENCE ABETA CLEARANCE BY IMMUNOTHERAPY
1
Y Peng1, M Maier2, L Jiang1, CA Lemere *
1. CND, Brigham & Women's Hospital, Boston, MA, USA
2. Department of Psychiatry, University of Zurich, Zurich, Switzerland
Complement proteins are part of the innate immune system and act as key mediators of
inflammation. Several components of the complement pathway are associated with neuritic
plaques in AD brain. To determine whether complement plays a role in AD pathogenesis and
clearance via immunotherapy, we crossbred complement C3-null mice with J20 APP tg mice.
Small increases in insoluble Abeta 40 and Abeta 42, but not soluble Abeta, in brain homogenates
were observed at 12 months of age in J20/C3-null mice relative to J20 complement-sufficient
mice. Plasma Abeta was slightly elevated, as were plaque deposition and gliosis. However, at 17
months, J20/C3-null mice had significantly less soluble Abeta and significantly more insoluble
Abeta40 and Abeta42 in brain homogenates and 51% more plasma Abeta compared to J20 mice.
Reactive microglia and astrocytes were increased as well. Hippocampal neurons in CA2/CA3 were
significantly reduced. No changes were observed in APP and alphaAPPs levels. Upon weekly
intranasal immunization with A1-40/42 and adjuvant LT(R192G) from 6-to-13 months of age,
APP/C3-/- mice generated Abeta antibodies of IgG2b and IgA isotypes that recognized the amino-
terminus of Abeta. These mice had significantly less insoluble AbetaX-42 in brain homogenates,
increased plasma Abeta, and significantly reduced plaque burden in hippocampus and cortex
compared to vehicle controls. Fibrillar Abeta plaques were unchanged but gliosis was reduced. No
T or B cells were detected in brain. Thus, it seems that C3 has a beneficial, neuroprotective role in
Abeta clearance/deposition in APP tg mice, however, complement C3 is not required for lowering
cerebral Abeta levels via Abeta immunotherapy.

DNA VACCINES FOR ALZHEIMER’S DISEASE WHICH COMBINE NON-SELF T-CELL EPITOPES

WITH MOLECULAR ADJUVANTS THAT UTILIZE THE INNATE IMMUNE SYSTEM TO DRIVE A TH2-
MEDIATED ANTIBODY RESPONSE
David H. Cribbs
University of California, Irvine
Active immunization of amyloid precursor protein (APP) transgenic mice with fibrillar beta-amyloid
(fAß) significantly attenuates Alzheimer’s-like amyloid plaque pathology. However, the first Aß-
immunotherapy trial in humans was halted during the Phase II stage when approximately 6% of
the participants developed meningoencephalitis. The vaccine for the clinical trial consisted of the
full-length fibrillar Aß and QS21, a powerful Th1 adjuvant. The cause of the meningoencephalitis
has not yet been definitively determined, however because some of the patients that developed
meningoencephalitis did not have detectable levels of anti-Aß antibodies, speculation has
focused on the composition of the immunogen and the type of adjuvant. Because many
autoimmune diseases have been linked to cell-mediated mechanisms and pro-inflammatory Th1-
mediated responses in particular, we have adopted alternative approaches to the immunogen
and the adjuvant for Aß-immunotherapy. First, to reduce the risk of an adverse T cell-mediated
immune response to Aß-immunotherapy we have replaced the Aß-self T cell epitope in the
immunogen with a synthetic T cell epitope, PADRE. Moreover, by increasing the number of N-
terminal Aß-B cell epitopes in the vaccine from one to three we have been able to significantly
enhance the anti-Aß antibody response. Secondly, by incorporating various “molecular adjuvants”
as fusion constructs with the actual immunogen into a vaccine that utilizes the innate immune
system to drive a Th2-mediated antibody response, we will actually induce an anti-inflammatory
cytokine response to the vaccine. A third component of our overall vaccine design is to utilize
DNA immunization, which offer many advantages over conventional protein/peptide methodology.
Previously, we demonstrated that gene gun immunization with a plasmid encoding only Aß 42
induced very low titers of anti-Aß antibodies in APP/Tg mice, and elicited unwanted anti-self Aß-
specific T cell responses. More recently, we demonstrated that DNA immunization with a plasmid
encoding IL4 fused with Aß dramatically enhanced antibody production over an Aß DNA construct
alone. Based on these initial findings we have pursued a series of novel second generation DNA
vaccines that have been specifically designed to further enhance the humoral immune response
to Aß-B cell epitope by taking advantage of the innate immune responses that can occur in the
skin. Thus, we have generated several new plasmid (p) constructs encoding p3Aß11-PADRE as the
immunogen, and either 1) macrophage-derived chemokine (MDC/CCL22), 2) beta-defensin-3
(BDF-3), or 3) three copies of C3d (3C3d), a cleavage fragment of complement C3 protein, as Th2
promoting molecular adjuvants. We have demonstrated that all three DNA vaccines induced
significant titers of anti-Aß antibodies, which were judged to functional based on multiple tests,
including blocking Aß assembly and binding to amyloid plaques. The efficacy of this approach in
various APP/Tg mice, where human Aß is a self-peptide, is currently under investigation. New pre-
clinical trials in APP/Tg mouse models may help to develop novel immunogen-adjuvant
configurations with the potential to avoid the adverse immune response that occurred in the first
clinical trial.

CLEARANCE OF AMYLOID BY ACTIVE IMMUNOTHERAPY HAS DIFFERENT CONSEQUENCES
THAN CLEARANCE BY AN NSAID
Dave Morgan, Donna Wilcock, Paul Jantzen and Marcia Gordon

University of South Florida
Passive immunotherapy has proven remarkably effective in clearing amyloid deposits from the
brains of APP transgenic mice. However, in old mice with well established deposits clearance is
associated with increased vascular amyloid deposits and microhemorrhage, conditions that might
have adverse effects if they occur in Alzheimer patients. Active immunization by vaccination has
not, thus far, been associated with these events. In this study we evaluated active immunization
of APP+PS1 transgenic mice from 10 until 20 mo of age on amyloid loads, vascular deposits and
microhemorrhage. At 10 mo APP+PS1 mice already have amyloid loads comparable to that of an
Alzheimer patient, although they are still young relative to their maximum lifespan of 28-30 mo. In
addition, mice were treated with the nitro-ferulo-flurbiprofen derivative, NCX-2216, which we had
previously shown could also reduce amyloid loads in these mice. A final treatment group received
both active immunization and NCX-2216. At the end of the study, vaccinated mice had
measurable anti-Aß antibody titers in spite of the fact that last immunization had been 2 mo prior
to tissue collection. Mice vaccinated against Aß had 25-30 percent reductions in Aß
immunohistochemistry in anterior cortex and hippocampus. NCX-2216 mice had 20% reductions
that were significant only in the hippocampus. Total Congo red staining was also significantly
reduced in both the hippocampus and frontal cortex by 25-30% with both treatments. However,
when Congophilic deposits were separated into vascular and parenchymal locations, the mice in
the vaccinated group had a 2 fold elevation of vascular deposits compared to untreated mice, in
spite of overall lowering of parenchymal deposits. NCX-2216 treated mice had no such changes in
vascular deposition. When sections were stained with Prussian blue to identify hemoglobin from
microhemorrhages, the vaccinated animals had a 3 fold increase over untreated mice. Again, no
increase was observed in mice treated with NCX-2216. Interestingly, in mice treated with both
vaccination plus NCX-2216, the results were identical to those observed with vaccination alone;
parenchymal deposits decreased, vascular deposits increased and microhemorrhage also
increased. In conclusion, these data indicate firstly that immunization of mice that already have
mature amyloid loads can lead to build up of vascular amyloid and increased risk of
microhemorrhage. Treating mice at ages when amyloid deposits are low appears to avoid this
consequence, suggesting this is associated with mobilization of pre-existing amyloid deposits. At
present it is unclear if these vascular events have pathological cognitive consequences, but
certainly the issue must be considered when conducting human trials with immunotherapy.
Secondly, high dose NSAID therapy can also reduce amyloid loads and to a comparable extent as
vaccination, but without vascular consequences. Moreover, the NSAID treatment does not a)
block any of the effects of vaccination (either clearance or vascular build up) or b) add with the
effects of vaccination in clearing Aß, arguing inflammation is not critically involved in either
process. Supported by AG 15490, AG 18478, AG 45711 and the Benjamin Trust.

Session VII: Aß-Peptide: Generation, Neurotoxicity &
SESSION VII: Aß-Peptide: Generation, Neurotoxicity & Treatment Strategies

Treatment Strategies
Chair: Christian Haass
The distinct protein aggregates that define Alzheimer's, Parkinson's, Huntington's and prion
diseases appear to cause these disorders. Small intermediates – soluble oligomers – in the
aggregation process can confer synaptic dysfunction, while large, insoluble deposits may serve as
reservoirs of the bioactive oligomers, which adversely affect synaptic structure and plasticity. In
this session we will review the molecular mechanisms of Aß-peptide generation (C. Haass), the
influence of Aß on synaptic function and memory (O. Arancio), and an Aß based therapeutic
strategy (anti-Aß vaccination; R. Nitsch).
Haass C, Selkoe DJ (2007) Soluble Oligomers in Neurodegeneration: Lessons from the

Alzheimer's Amyloid ß-peptide. Nature Reviews Molecular Cell Biology, 2, 101 - 112.
ALZHEIMER’S DISEASE: DEFEATING IT AT THE SYNAPSE
Ottavio Arancio
Columbia University
The Aβ peptide is likely to play a central role in Alzheimer’s disease. The production of elevated
amounts of Aβ interferes with the activation of important molecules that are needed for memory,
including the memory related molecule CREB. Drugs that up-regulate the phosphorylation of CREB
produce an immediate and long-lasting beneficial effect on abnormal synaptic function and
memory following elevation of the peptide. However, Aβ is normally produced in the brain
throughout life. Nevertheless, it is not known whether Aβ has a physiological role in the brain. Our
lack of understanding of the physiological role of Aβ may present important issues when
designing effective and safe approaches to Alzheimer therapy. We have now obtained compelling
data demonstrating that Aβ itself is a critical positive-modulator of synaptic plasticity and memory
at physiological concentrations within the normal brain. The presence of the -7 nicotinic receptor
is necessary for this effect to occur. Indeed, and paradoxically, the use of drugs that mimic Aβ
structure, or targeted to the receptor(s) on which Aβ acts under normal physiological conditions,
or even of Aβ itself or Aβ derivatives might serve to enhance synaptic plasticity and memory at
appropriate concentrations.

IMMUNOTHERAPY AGAINST BETA-AMYLOID IN ALZHEIMER’S DISEASE
SESSION VII Aß-Peptide: Generation, Neurotoxicity & Treatment Strategies
Roger M. Nitsch and Christoph Hock
University of Zurich
Immunotherapies targeting brain beta-amyloid are currently tested for safety and efficacy in
patients with Alzheimer's disease (AD). Initial attempts with active Abeta immunization were
halted because of the development of meningoencephalitis in a subgroup of patients, but follow-
up studies established that Abeta immunization induced chronic elevations of antibodies against
beta-amyloid. These are transported across the blood brain barrier into brain tissue and
cerebrospinal fluid. In patients who had developed such antibodies, cognitive decline was slowed,
brain beta-amyloid load was greatly reduced, and shrinkage of hippocampus volume was
prevented. These initial beneficial clinical effects were independent of the prior occurrence of
meningoencephalitis. Together, these pilot data suggest that chronically elevated titers of
antibodies against beta-amyloid in AD patients could be safe and potentially efficacious in
reducing the decline of cognitive functions, hippocampus tissue loss and beta-amyloid-related
neuropathology.
A NOVEL HIGH THROUGHPUT SCREEN FOR Aβ POLYMERIZATION INHIBITORS
Lars Tjernberg*, Hiroyoshi Kakuyama, Bengt Winblad and Niklas Bark

The polymerization of the amyloid β-peptide (Aβ) into fibrillar deposits in the human brain appears
to be a pathogenic event in Alzheimer’s disease. One possible treatment strategy would thus be to
attenuate Aβ polymerization. Most of the commonly used methods for studying Aβ-polymerization
are unsuitable for high throughput screening (HTS), and no comprehensive investigation of large
compound libraries have been reported. To enable HTS for Aβ-polymerization inhibitors, we have
developed a fluorescence resonance energy transfer (FRET) assay. FRET is a phenomena where a
fluorescent donor molecule in an excited state instead of emitting light excites an acceptor
molecule. This process is strongly dependent on the distance between the donor and the
acceptor, and can be used to measure the distance between these. Residues 16-20 in Aβ are
important for Aβ-Aβ interactions, and compounds binding to this region attenuate Aβ
polymerization. We constructed a peptide with two Aβ-Aβ binding motifs connected by a turn-motif
and added a FRET pair at the N- and C-terminus. We speculated that this peptide would be in a β-
hairpin conformation with a short distance between donor and acceptor, resulting in a strong
FRET signal. The peptide showed strong FRET, which was abolished under denaturing conditions.
Different known inhibitors were tested, and curcumin was found to decrease FRET in a dose
dependent manner. CD-spectroscopy showed that the peptide to a large extent was present in a
β-sheet conformation, which decreased after the addition of curcumin. Thus, compounds binding
to the peptide disrupt the β-hairpin conformation and increases the distance between donor and
acceptor, thereby decreasing FRET. A peptide concentration of 1 µM gives a strong FRET signal in
a microwell plate setting, and the incubation times are short. We conclude that the assay works
as intended, and it is presently used for screening a large compound library.

REGULATED INTRAMEMBRANE PROTEOLYSIS IN ALZHEIMER'S DISEASE
SESSION VII: Aß-Peptide: Generation, Neurotoxicity & Treatment Strategies

Christian Haass
Ludwig-Maximilians-University
Amyloid β-peptide (Aβ) generation has turned out to be just one example of a general
physiological mechanism now known as regulated intramembrane proteolyis (RIP). In a principal
version of this process, membrane proteins first undergo a regulated shedding of their
ectodomains by membrane-anchored proteases referred to as secretases or sheddases, releasing
the large luminal domains into extracellular fluids. The membrane-retained stubs can then be
cleaved within their transmembrane domains (TMDs) to release small hydrophobic peptides (e.g.,
Aβ in the case of APP) into the extracellular space and the intracellular domains (ICDs) into the
cytoplasm. The free ICDs may have specific functions in the cytosol, including the activation of
nuclear signaling pathways, as in the case of the Notch ICD. In the case of APP and certain other
RIP substrates, ectodomain shedding can be effected by either of two distinct membrane-
anchored proteases, α-secretase (believed to be one or more members of the ADAM family of
metalloproteases) or β-secretase (also called β-site APP cleaving enzyme or BACE). The
membrane associated stub created by BACE cleavage can then undergo an intramembrane
scission mediated by a unique protease complex, γ-secretase, that is composed of four proteins.
These are presenilin-1 or 2 (PS1/PS2), Nicastrin, APH-1, and PEN-2. All four proteins are both
necessary and sufficient to reconstitute γ-secretase activity. At least in the case of its two most
well studied substrates, APP and Notch, γ-secretase can carry out multiple intramembrane
cleavages.

Session VIII: The Role of BACE Inhibition in Alzheimer's
Disease
Chair: Stefan Berg
THE β-SECRETASE BACE1 IN ALZHEIMER’S DISEASE
R. Vassar, J. Zhao, T. O’Connor, Y. Fu, R. Velliquette, E. Maus, B. Hitt, M. Yasvoina, P. Shao, S.

Logan, R. Berry, and L. Binder
Northwestern University Feinberg School of Medicine
BACE1 (beta-secretase) initiates cleavage of Abeta from APP and is a promising therapeutic target
for Alzheimer’s disease (AD). BACE1 levels are elevated in AD and may increase Abeta, but the
SESSION VIII: The Role of BACE Inhibition in AD
cause of BACE1 elevation is unknown. Interestingly, glucose metabolism is reduced in AD,

suggesting impaired energy metabolism may be pathogenic. Here, we investigated whether
energy inhibition increases BACE1 and Abeta in APP transgenic mice. We also examined the role
of amyloid deposition in BACE1 elevation. First, we used drugs (e.g., insulin, 2-deoxyglucose, 3-
nitropropionic acid) to induce acute and chronic energy inhibition in APP transgenic (Tg2576)
mice. We observed that BACE1 and Abeta levels increased ~2-fold in mice treated acutely (1
injection) or chronically (3 injections/week, 3 months). Chronically treated Tg2576 mice had
increased plaque numbers, supporting the hypothesis that energy inhibition may contribute to AD.
Next, we made a mono-specific anti-BACE1 monoclonal antibody in BACE1-/- mice and with it
determined that BACE1 levels were increased ~2-fold in human AD and APP transgenic brains. In
our new rapid amyloid deposition model, 5XFAD mice, the BACE1 increase started at 4 months
and rose in parallel with amyloid burden, demonstrating that BACE1 elevation occurred early
rather than late, excluding end-stage neurodegeneration as a cause. Increased BACE1 levels were
associated with amyloid plaques in 5XFAD, Tg2576, and AD brains. Immunofluorescence
microscopy showed elevated BACE1 surrounding Aβ42-positive amyloid plaque cores in neuron-
derived structures. We conclude that energy impairment and amyloid deposits cause BACE1
levels to increase, potentially causing a positive feedback loop driving Abeta production in AD.
THE DISCOVERY OF NON TRANSITION-STATE ISOSTERE BACE INHIBITORS
Hemaka A. Rajapakse
Merck Research Laboratories
Incorporation of traditional aspartyl protease transition-state isosteres such as statines and

hydroxyethylamines into peptidomimetic scaffolds has resulted in the discovery of potent BACE
inhibitors. Their therapeutic use to treat Alzheimer's disease has, however, been limited by the
challenge of combining potency and adequate physical properties of such inhibitors to permeate
the CNS. The discovery of a novel aspartyl protease binding motif and its incorporation into potent
BACE inhibitors displaying improved physical properties will be presented. The binding of this
motif in the catalytic site of BACE and its impact on the enzyme's topology will be described and
illustrated by several crystal structures.

NOVEL GSK3 INHIBITORS FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES
SESSION VIII: The Role of BACE Inhibition in AD

S. Berg, R. Bhat, S. Hellberg
AstraZeneca AB
Glycogen synthase kinase 3 (GSK3) was initially described as an enzyme involved in glycogen
metabolism but has now emerged as one of the most attractive targets for development of new
drugs for neurodegenerative diseases. Selective GSK3 inhibitors which inhibit
hyperphosphorylation of the microtubule associated protein tau may represent an effective
pharmacotherapy in tauopathies such Alzheimer’s Disease as such an approach would slow down
the progression of neurofibrillary pathology. We have identified a series of small molecule
inhibitors of GSK3 that have the potential to interfere with tau phosphorylation and neurofibrillary
pathology. In particular, a series of small molecule inhibitors known as pyrazines were identified
from a high throughput screening campaign. These compounds have been optimized to result in
highly potent GSK3 inhibitors. The inhibitors are ATP competitive analogues and exhibit excellent
selectivity over related kinases. Furthermore, they display good cellular efficacy. These analogues
provide viable GSK3 inhibitors in the discovery of new therapies for the treatment of
neurodegenerative diseases.

Session IX: Genetic Implication of Tau in Alzheimer's
Pathogenesis
Chair: John Hardy
THE ROLE OF PROGRANULIN IN DEMENTIA
SESSION IX: Genetic Implication of Tau in AD Pathogenesis
R. Rademakers1, M. Baker1, I.R. Mackenzie 2A, C. Andorfer 1, J. Eriksen 1, J. Gass 1, A. Cannon 1, B.

Boeve 3, J. Adamson 1, R. Crook 1, R. Petersen 3, K. Josephs 3, N. Graff-Radford 4, D. Dickson 1, Z.
Wzsolek 4, H. Feldman 2B, D. Knopman 3 and M. Hutton 1
1Department of Neuroscience, Mayo Clinic College of Medicine; 2 (A)Department of Pathology, (B) Division
of Neurology, University of British Columbia; 3 Department of Neurology, Mayo Clinic College of Medicine; 4
Department of Neurology, Mayo Clinic College of Medicine
We recently demonstrated that null mutations in the gene encoding progranulin (PGRN) cause
tau-negative frontotemporal dementia (FTD) linked to chromosome 17. PGRN encodes a 68.5
kDa secreted mitogenic factor, thought to be involved in the regulation of multiple processes
including development, wound repair and inflammation. Patients with mutations in PGRN are
characterized by the formation of tau-negative, ubiquitin-positive neuronal cytoplasmic and
intranuclear inclusions recently shown to contain TAR DNA binding protein (TDP43). This
neuropathological phenotype distinguishes these patients from FTD patients with mutations in the
gene encoding the microtubule associated protein tau (MAPT) given that these individuals
inevitably have tau-positive neurofibrillary lesions and do not develop consistent TDP-43 inclusion
pathology.
In an extensive mutation screen we identified PGRN mutations in 10% of our Mayo Clinic FTLD
series and in 23% of patients with a positive family history of dementia. Clinical examination of
patients revealed highly variable onset ages with language dysfunction as a common presenting
symptom. The role of progranulin as a genetic risk factor for FTD related neurodegenerative
disorders such as Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS) is currently
being studied.
All pathogenic mutations in PGRN identified to date create functional null alleles implying that
they cause disease by producing a deficiency of progranulin. However, the role of progranulin in
neuronal survival and the explanation for why partial loss of this factor causes adult-onset
neurodegenerative disease remains uncertain. What is known is that progranulin has been
consistently implicated in the mechanism of wound repair and that progranulin is upregulated in
activated microglia in response to the development of multiple neurodegenerative diseases
including AD. In addition, the dramatic accumulation of progranulin in dystrophic neurites
surrounding senile plaques suggests that progranulin is involved in the localized brain response
to amyloid deposition in AD. However, whether progranulin is critical to the mechanism of brain
repair and whether deficiency in this process is linked to the process of neurodegeneration
remains to be determined.
NOS2 DELETION PROMOTES MULTIPLE PATHOLOGIES IN A NEW MOUSE MODEL OF

ALZHEIMER’S DISEASE
M. P. Vitek, D. A. Wink, Q. Xu, V. Cantillana, H. Dawson, M. L. Preveti, B. Weinberg, W. E. Van

Nostrand and C. A. Colton
Duke University Medical Center, National Cancer Institute, and Stony Brook University
Alzheimer’s disease (AD) is characterized by two primary pathologies: amyloid plaques and
neurofibrillary tangles (NFTs). In addition, neuronal loss is prominent in a variety of brain regions

and the vast majority of AD patients also display cerebrovascular deposits of fibrillar Amyloid Beta
SESSION IX: Genetic Implication of Tau in AD Pathogenesis

Peptide (Abeta). The amyloid hypothesis suggests that deposition of fibrillar Abeta can lead to
these other characteristics of AD and thus, can initiate the disease process. Transgenic animals
overexpressing Abeta peptide, however, are largely devoid of all characteristics of AD except for
deposition of fibrillar Abeta as amyloid plaques and/or cerebrovascular amyloid. To address the
relationship between deposition of Abeta, the formation of NFTs and neuronal loss, we
investigated the role of nitric oxide (NO) as part of the inflammatory response that is found in AD
brains and the brains of APP transgenic mice. The amount of NO produced by human iNOS is
significantly less than the amount of NO produced by mouse iNOS. By mating an APP trangenic
mouse, Tg2576, to a NOS2 knockout, we created a double transgenic mouse (hAPP/NOS2-/-) that
results in fibrillar Abeta deposition, pathological hyperphosphorylation of mouse tau,
redistribution of phospho-mouse tau into the somatodendritic compartment of neurons, mouse
tau aggregation and fluorojade positive degenerating neurons. We recently mated the SwDI
transgenic APP mouse to a NOS2 knockout and created a second double transgenic mouse
(SwDI-APP/NOS2-/-) that also results in fibrillar Abeta deposition, pathological
hyperphosphorylation of mouse tau and fluorojade positive degenerating neurons. Both of these
mice display enhanced expression of activated caspase-3 that we suggest can cleave mouse tau
and thereby promote pathological tau phosphorylation and aggregation. These data suggest that
Nitric Oxide may play a critical role in controlling the formation of AD-like pathology in novel mouse
models of Alzheimer’s disease.
GENETIC DISSECTION OF THE ETIOLOGIES AND PATHOGENESES OF ALZHEIMER’S AND

PARKINSON’S DISEASES AND RELATED DISORDERS
John Hardy
National Institute on Aging, National Institutes of Health
Genetic analyses have implicated mutations in the APP gene as one cause of early onset,
autosomal dominant, Alzheimer’s disease. These mutations, like mutations in the presenilin
genes, alter the amount of the peptide, A42 produced during APP processing. Genetic analysis
of a recent genome screen for late onset Alzheimer’s disease, has implicated the APP locus as
risk factor locus, for late onset disease although frank mutations have not been found: this
suggests that genetic variability in APP expression contributes to the risk of this form of the
disorder: not surprising, perhaps, given the longstanding association between trisomy 21 and AD.
Similarly, mutations in the tau gene cause autosomal dominant tangle disease (FTDP-17), and
genetic variation at the tau locus, but not coding changes, is associated with the sporadic tangle
diseases, progressive supranuclear palsy and corticobasal degeneration: this suggests that
genetic variability in either tau expression or in tau splicing contributes to the risk of these
diseases. I will present data showing that the precisely delineated risk MAPT haplotype drives
higher levels of expression of tau and a greater proportion of the 4 repeat isoform. Finally,
mutations in the -synuclein gene cause autosomal dominant Lewy body disease, and genetic
variability (haplotypic association) at the -synuclein locus contributes to sporadic disease: our
recent demonstration that one cause of autosomal dominant disease is a triplication of the -
synuclein locus, indicates that the most likely explanation of this observation is that this
haplotypic association reflects the fact that genetic variability in the control of -synuclein
contributes to disease risk. These observations have in common support for the notion that these
common diseases are initiated by overexpression of key pathogenic proteins which are close to
their threshold of solubility.

Session X: Degradation, Aggregation and
Phosphorylation: Pathogenic Potential and Therapeutic
Opportunities in Alzheimer's Disease
Chair: Karen Duff
SESSION X: Degradation, Aggregation & Phosphorylation
Alzheimer’s disease is characterized by two pathologies – plaque and tangle formation, but many
pathways have been implicated in the formation of those lesions. Three pathways will be
examined. Dr Chiosis will discuss the abnormal turnover of tau through the Hsp90 system,
including the effects of an inhibitor of Hsp90 on tauopathy in mice. Dr. Duff will discuss the effect
of phosphorylation modulators, especially GSK and Cdk5 on tauopathy and amyloidogenesis and
Dr. Kuret will discuss the kinetics of tau fibrillarization and modulators of this process.
ROLES OF HEAT SHOCK PROTEIN 90 IN MAINTAINING AND FACILITATING THE DEGENERATIVE

PHENOTYPE IN TAUOPATHY
Gabriela Chiosis
Sloan-Kettering Institute
Neurodegeneration is a result of multiple dysregulatory events, a lengthy multi-step process

manifested by an accrual of mutant variants, and abnormal expression, post-translational
modification, and processing of certain proteins. The accumulation of these dysregulated
processes requires a mechanism that maintains their functional stability and allows for the
evolution of the neurodegenerative phenotype. In malignant cells, capacity to buffer
transformation has been attributed to heat shock protein 90 (Hsp90). While normal proteins
seem to require limited assistance from the chaperone, their mutated or aberrant counterparts
appear to be highly dependent on Hsp90. Whereas enhanced Hsp90 affinity for mutated or
functionally deregulated client proteins has been observed for several oncoproteins, it is not
known whether Hsp90 plays a similar role in regulating aberrant neuronal proteins, and thus
maintains and facilitates the transformed phenotype in neurodegenerative diseases. Tauopathies
are neurodegenerative diseases characterized by aberrant phosphorylation and/or expression of
Tau protein leading to a time-dependent accumulation of Tau aggregates and subsequent
neuronal death. We show that the stability of both p35, a neuronal protein that activates cdk5
through complex formation leading to aberrant Tau phosphorylation, and that of mutant but not
WT Tau protein are maintained in tauopathies by Hsp90. Inhibition of Hsp90 in both cellular and
mouse models of tauopathies by small molecules developed by our laboratory leads to reduction
of the pathogenic activity of these proteins and results in elimination of aggregated Tau.
The results identify important roles played by Hsp90 in maintaining and facilitating the
degenerative phenotype in these diseases, and provide a common principle governing cancer and
neurodegenerative diseases.

MECHANISMS OF TAU AGGREGATION: HOW DO POST-TRANSLATIONAL MODIFICATIONS AND
SESSION X: Degradation, Aggregation & Phosphorylation

TAU SPLICING MUTATIONS PROMOTE NEUROFIBRILLARY LESION FORMATION?
Jeff Kuret
Ohio State University College of Medicine
Filamentous inclusions composed of the microtubule associated protein tau are found in AD and
other tauopathic neurodegenerative diseases. To clarify the mechanism through which tau
aggregates from monomer, the fibrillization of recombinant full-length four-repeat human tau was
examined in vitro as a function of time and submicromolar tau concentrations using electron
microscopy methods and a small-molecule inducer of aggregation, thiazin red. Data were then fit
to a simple homogeneous nucleation model with rate constant constraints established from
filament dissociation rate, critical concentration, and mass-per-unit length measurements. The
results suggest that the rate-limiting step in the fibrillization of assembly competent tau
conformations is formation of a dimeric nucleus, which normally is unstable under sulfhydryl
reducing conditions, and that filament elongation proceeds via addition of tau monomers to
nascent filament ends. Extending the data to include all six naturally occurring isoforms yielded
estimates of the contribution of each tau exon to aggregation behavior. The presence of Exon 10
decreased critical concentration ca. 6-fold, and also speeded nucleation rate. The former effect
synergized with Exon 2, so that the presence of both Exons 2 and 10 decreased critical
concentration by a full order of magnitude. The data illustrate how post-translational
modifications, including those arising from oxidative stress, as well as changes in isoform
composition, can promote tau fibrillization in disease by modulating key rate-limiting steps.
ABERRANT PHOSPHORYLATION AS A PATHOGENIC MECHANISM IN ALZHEIMER'S DISEASE
Karen Duff
Columbia University/NYS Psychiatric Institute
Alzheimer’s disease is characterized by the presence in the brain of Aβ containing plaques, and
tangles containing filamentous, hyperphosphorylated tau. Hyperphosphorylation has been cited
as a pathogenic mechanism in the production of pathogenic tau and tangle formation, and we
have shown that kinases cdk5 and GSK3 can impact this process (Noble et al, Neuron 2003,
PNAS 2005). These kinases are also implicated in Aβ production, and our recent work has shown
that Aβ levels are increased in mice overexpressing the cdk5 activator p25. Enhanced cdk5
activity in these mice, and in cell culture models correlates with increased levels of APP
metabolites including the amyloidogenic Aβ peptides. The APP processing enzyme BACE1, is also
increased in response to elevated p25, and it accumulates in early endosome compartments
which are known to favor APP processing. Deletion analysis shows that a region of the BACE1
promoter is responsive to p25/cdk5 suggesting that upregulation of BACE through transcriptional
control leads to increased Aβ levels, which may be relevant for AD pathogenesis.

POSTERS
LIFELONG CALCIUM DISRUPTIONS IN ALZHEIMER’S DISEASE MICE: NORMAL AGING VS.

PATHOLOGICAL SIGNALING CHANGES IN NEURONS
Grace E. Stutzmann*.1, Ian Smith 2, Antonella Caccamo2, Salvatore Oddo 2, Ian Parker 2, Frank
LaFerla2
1 Rosalind Franklin University / The Chicago Medical School, 2 University of California, Irvine
Proper maintenance of intracellular calcium signals is critical for neuronal functioning, both in the
short term and over an organism’s lifetime. Exaggerated ER calcium responses are associated
with Alzheimer disease mutations, however the mechanism is unclear. Here, our objective is to
determine the receptor-mediated dynamics that contribute to exaggerated ER-calcium release,
and determine if this is distinct from alterations associated with normal aging. By use of 2-photon
calcium imaging, whole-cell electrophysiological recordings, and flash photolysis of caged
compounds in cortical brain slices we distinguished between IP3R- and RyR-mediated calcium
components in non-transgenic controls (Non-Tg), and three separate AD-mouse models (3xTg-AD,
PS1KI, and APP/Tau). Our results demonstrate exaggerated (>4-fold) ER calcium signals in the
PS1KI and 3xTg-AD mice relative to NonTg mice. The PS1 mutation was the predominant
‘calciopathic’ factor, as responses in 3xTg-AD mice were similar to PS1KI mice, and APPSweTauP301L
mice were not different from controls. We also quantified patterns of calcium signaling
dysregulation in 6 week, 6 month, and 1.5 year old mice. The effects of AD trangenes across the
different age points demonstrate marked increases in ER-calcium release in the PS1KI and 3xTg-
AD neurons relative to NonTg at 6 weeks.. At 6 months, the PS1KI and 3xTg-AD mice show
similarly enhanced ER-calcium responses relative to the NonTg neurons (2 fold), while at 1.5
years, the PS1KI mice show up to a 1.5-fold enhancement of ER-calcium responses and the 3xTg-
AD more than a 2-fold enhancement. The exaggerated signals in 3xTg-AD and PS1KI mice
resulted primarily from enhanced RyR signaling, and correspond with increased RyR expression
levels. We further conclude that the PS1 mutations contribute to long-term calcium signaling
dysregulations which are not a part of normal aging. In addition, the deposition of Aβ plaques and
POSTER PRESENTATIONS
tangles seen in older 3xTg-AD mice may further contribute to calcium signaling alterations.
UNFOLDED p53 PROTEIN AS A PERIPHERAL AD MARKER
C. Lanni, 1 M. Racchi, 1 G. Mazzini, 3 A. Ranzenigo,4 R. Polotti, 4 S. Govoni, 1 M. Memo, 2 D.

Uberti
University of Pavia; 2 University of Brescia; 3 University of Pavia; 4 Ospedale Generale di zona S.
Orsola FBF, Brescia
Early diagnosis of Alzheimer's disease is an important first step in management. The development
of peripheral marker can be useful for identifying vulnerable population and monitoring the
progression of the disease. The biological markers can be classified as primary (specific), such as
beta amyloid, or secondary to the disease, or they can simply be epiphenomenal in nature.
In research of secondary markers we recently found an intriguing correlation between p53 and
AD. In particular, we demonstrated that fibroblasts from sporadic AD patients specifically express
an unfolded and detectable conformational state of p53 that allows to differentiate them from

fibroblasts of age-matched non-AD subjects. In this study we enrolled more than 150 subjects and
tested the content of unfolded p53 in AD, non AD and subjects affected by other dementia, by
using a rapid and easy cytofluorimetric approach. We found that peripheral blood cells from AD
specifically expressed increased levels of unfolded p53 in comparison with non AD subjects. The
biological markers were specific for AD because low levels of unfolded p53 was found in the blood
of subjects affected by other dementia. Moreover a statistically significant correlation was
observed when the expression of unfolded p53 and the age of both control subjects and AD
patients were considered thus demonstrating that altered p53 is an age-dependent factor. In
order to evaluate the diagnostic performance of unfolded p53 as an AD trait marker, we
calculated sensitivity and specificity within different age intervals and we found that these values
were more significant in subjects up to 70 years of age compared with the corresponding values
for individuals older than 70 years. Within this specific age interval (< 70 years), we worked out a
sensitivity of 90% to discriminate AD patients from non demented aged individuals at a specificity
value of 77%.
CHARACTERISATION OF CELL-PERMEABLE BACE-1 INHIBITORS IN PRIMARY NEURONS
Christiane Volbracht*, Tenna Juul Schrøder, Lars Østergaard Pedersen, Søren Christensen,
Pekka Kallunki, Christian Thomsen
H. Lundbeck A/S
The primary factor driving pathogenesis in Alzheimers’ disease (AD) according to the amyloid
hypothesis is the accumulation of Amyloid β-peptide (Aβ) in the brain. Aβ is generated by
sequential processing of amyloid precursor protein (APP) by β and γ-secretases. BACE-1 β-site APP
Cleaving Enzyme 1) has been identified as the enzyme responsible for the β-secretase activity and
is the key rate-limiting enzyme that initiates the formation of Aβ. BACE-1 appears as promising
therapeutic target for amyloid-lowering strategies in AD.
Here, we report the development of cellular assays in primary neuronal cultures and
characterization of cell-permeable BACE-1 inhibitors. We transfected murine cerebellar granule
cell (CGC) cultures with human APP using an electroporation-based method (Nucleofection™).
Endogenous murine secretases cleaved human APP and Aβ1-40/1-42 was secreted into the
culture medium. In addition, we established cortical cultures and CGC from wildtype mice to test
cell-permeable secretase inhibitors. In neurons, BACE-1 inhibitors displayed a dose-dependent
reduction of Aβ generation with an IC50 in the low nM range. Furthermore, we observed reduction
in sAPPβ (N-terminal product of APP following BACE-1 cleavage) and elevation in APP. In contrast
to γ-secretase inhibitors, BACE-1 inhibitors increased the amount of sAPPα (N-terminal product of
APP following α-secretase cleavage). Additionally to the use of secretase inhibitors, we validated
these neuronal models genetically by knock-down of BACE-1 expression by siRNA. We observed
50-70% inhibition of Aβ1-40 formation with murine specific BACE-1 siRNA in concert with
reduction of the sAPPβ fragment. Robust reduction of Aβ; in mice brain homogenates was
observed upon BACE-1 and γ-secretase inhibition within 3h. These studies confirm
pharmacologically, that BACE-1 plays the predominant role in the β;-site cleavage of APP and that
inhibition of β-secretase activity could potentially redirect APP-processing via the non-
amyloidogenic α-secretase pathway.

PREVENTION OF FIBRIL FORMATION AND ABETA-MEDIATED NEUROTOXICITY BY ANTI-D-
PEPTIDE ANTIBODIES
M. Yu*, F. Ouellet, D. Stéa, M. Azzi, G. Sebastiani, D. Delorme, B. D. Greenberg

Neurochem Inc.
Immunization with Abeta1-42 has been shown to reduce cerebral amyloid deposition in
transgenic mouse models of brain amyloidosis by the induction of both adaptive and innate
immunity. In particular, the adaptive immune response is mediated by Abeta-specific antibodies.
It is possible to reduce Abeta fibril formation and to overcome inflammation of an amyloid vaccine
by using short fragments of Abeta. We have produced antibodies against D- and L-peptides
derived from the core Abeta region, which is central to fibrillogenesis and cellular interactions. By
this, we attempt to bind soluble rather than fibrillar Abeta, and prevent conformational transitions
that lead to the accumulation of toxic oligomeric forms, proto-fibrils, and plaques. Such polyclonal
antibodies were demonstrated in vitro to prevent fibril formation as measured by a fluorescence
assay employing Thioflavin T (ThT), and protect against Abeta-induced neurotoxicity, where anti-D-
peptide polyclonal antibodies were more active than the anti-L-peptide counterpart. To further
characterize and confirm these effects, 15 monoclonal antibodies (MAbs) were tested for their
ability to prevent beta-amyloid formation. Three MAbs demonstrated promising anti-amyloid
activity in vitro delaying fibril formation in the ThT assay and inhibiting Abeta-induced DNA
fragmentation/condensation in primary rat neurons and human neuroblastoma SH-SY5Y cells.
Moreover, co-treament of Abeta with each MAb decreased Abeta -induced toxicity both by
reducing caspase 3/7 activity and by increasing cell viability as measured by WST-1 assay. Taken
together, these data demonstrate that anti-D-peptide antibodies from the core region of Abeta
molecule prevent fibril formation and Abeta-mediated neurotoxicity in vitro and raise the potential
for in vivo efficacy.
WHERE EXACTLY IN THE CELL DO THE SECRETASES CLEAVE? TARGETING THE LOCATION FOR
THERAPY
Lawrence Rajendran, Cornelia Schroeder, Georg Schlechtingen, Sebastian Weidlich, Patrick

Keller, Gary Jennings, Hans-Joachim Knoelker, Kai Simons
Max Planck Institute of Molecular Cell Biology and Genetics, Technical University and JadoLabs
GmbH
Evidence exists to support the idea that the lipid microdomains in the memrbane and the
endocytic system play a crucial role in the pathogenesis of AD. However, two important questions
remain: the identity of the subcellular compartment where the secretases cleave and the
mechanism by which the intracellularly generated Abeta is released into the extracellular milieu.
Here we show that these cleavages require the localization of the beta secretaase in membrane
microdomains (lipid rafts) and their entry into the early endosomes. Targeting a beta-secretase
inhibitor to lipid raft domains in plasma membrane inhibited the secretase more efficiently than
its soluble counterpart suggesting on one hand, a novel therapeutic strategy and on the other
hand, that the site of beta-cleavage being endosomes and not plasma membrane.

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ABSTRACTS 13

SESSION I: MODULATORS OF AD NEUROPATHOLOGY 14

SESSION II: DISEASE MODIFICATION STRATEGIES FOR AD 16

SESSION III: GAMMA-SECRETASE: FUNCTIONALITY AND PHARMACOLOGY 18

SESSION IV: GENETICS AND TREATMENT STRATEGIES FOR ALZHEIMER'S DISEASE 20

SESSION V: IMAGING AND MODULATION OF NEUROPATHOLGIES 22

Page 10 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION VI: IMMUNOTHERAPY FOR ALZHEIMER'S DISEASE 24

SESSION VII: Aß-PEPTIDE: GENERATION, NEUROTOXICITY & TREATMENT STRATEGIES 27

SESSION VIII: THE ROLE OF BACE INHIBITION IN ALZHEIMER'S DISEASE 30

SESSION IX: GENETIC IMPLICATION OF TAU IN ALZHEIMER'S PATHOGENESIS 32

SESSION X: DEGRADATION, AGGREGATION AND PHOSPHORYLATION: PATHOGENIC POTENTIAL AND

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 11

Page 12 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 13

ß-AMYLOID DEGRADATION: THE INSIDE STORY

Malcolm A. Leissring*, Ji Zhao, Lilin Li, Qun Lu & Malwina Huzarska

proteases may contribute differentially to different aspects of AD pathogenesis by virtue of

CEREBRAL MICROVASCULAR AMYLOID: NEUROINFLAMMATION AND COGNITIVE DEFICITS

Cerebral microvascular amyloid ß protein (Aß) deposition and associated neuroinflammation is

Page 14 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION I: Modulators of AD Neuropathology

Next, we investigated the effect of the anti-inflammatory drug minocycline on Aß accumulation,

REVERSING AGE-DEPENDENT MEMORY IMPAIRMENTS IN A TRANSGENIC MODEL OF

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 15

GSK3 INHIBITORS FOR ALZHEIMER'S DISEASE

TRANSLATIONAL BIOMARKERS FOR DISEASE MODIFICATION IN ALZHEIMER'S DISEASE

Page 16 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION II: Disease Modification Strategies for AD

PRX-07034 and PRX-03140 - NOVEL, SELECTIVE AGENTS TARGETING SEROTONIN RECEPTORS

STABLE TRIMERIC A-BETA PEPTIDES: POTENTIAL VACCINE IMMUNOGENS TO PREVENT ADDL-

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 17

Session III: Gamma-secretase: Functionality and

-SECRETASE: PAST, PRESENT, FUTURE

Alexandra Tolia and Bart De Strooper

Page 18 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION III: Gamma-secretase: Functionality and Pharmacology

LY450139: TESTING THE AMYLOID CASCADE HYPOTHESIS IN MAN

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 19

CLINICAL GENETICS IN ALZHEIMER'S DISEASE

CSF BIOMARKERS IN ALZHEIMER DISEASE CLINICAL TRIALS

Alzheimer disease (AD) is multifactorial, heterogeneous and involves different disease

Page 20 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION IV: Genetics and Treatment Strategies for AD

Memantine, an uncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors, is approved

TREATMENT STRATEGIES IN SEVERE DEMENTIA

In developed countries, where elderly patients constitute an escalating proportion of the

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 21

MECHANISMS OF APOE CONTRIBUTION TO ALZHEIMER'S DISEASE

A VIEW INTO THE ALZHEIMER'S BRAIN WITH OPTICAL IMAGING

Page 22 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION V: Imaging and Modulation of Neuropathologies

CHOLESTEROL MODIFYING STRATEGIES FOR ALZHEIMER'S DISEASE

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 23

preparations and mechanisms of antibody mediated amyloid clearance. Development of

COMPLEMENT C3 DEFICIENCY LEADS TO INCREASED PLAQUE BURDEN AND NEURONAL LOSS

Page 24 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION VI: Immunotherapy for Alzheimer's Disease

Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery Page 25

Dave Morgan, Donna Wilcock, Paul Jantzen and Marcia Gordon

Page 26 Alzheimer’s Disease: From Molecular Mechanisms to Drug Discovery

SESSION VII: Aß-Peptide: Generation, Neurotoxicity & Treatment Strategies

Haass C, Selkoe DJ (2007) Soluble Oligomers in Neurodegeneration: Lessons from the

ALZHEIMER’S DISEASE: DEFEATING IT AT THE SYNAPSE