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EMBO reports 4, 8, 747–752 (2003)
doi:10.1038/sj.embor.embor905
AOP Published online: 25 July 2003
Attack on amyloid
International Titisee Conference on Alzheimer's and Parkinson's
Disease: From Basic Science to Therapeutic Treatment
Philipp J. Kahle1 & Bart De Strooper2
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1 Laboratory of Alzheimer's and Parkinson's Disease
Research, Department of Biochemistry, Ludwig Maximilians University,
Schillerstrasse 44, 80336 Munich,
Germany Tel: +49 89 5996 480; Fax: +49 89 5996 415;
e-mail: pkahle@pbm.med.uni-muenchen.de
2 Center for Human Genetics, Catholic University of
Leuven and Flemish Institute of Biotechnology, Herestraat
49, 3000 Leuven, Belgium Tel: +32 16
346227; Fax: +32 16 347181;
e-mail: bart.destrooper@med.kuleuven.ac.be
Received 7 May 2003; Accepted 24 June 2003; Published online 25 July 2003.
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Almost 100 years after Alois Alzheimer saw his first patient with
the complaint of "having lost herself" and his subsequent
neuropathological description of what is now known as Alzheimer's disease,
Christian Haass and Roger Nitsch invited a panel of international opinion
leaders to the romantic Lake Titisee in the German Black Forest. This meeting,
which took place during 19–23 March 2003, was the 87th International
Titisee Conference, sponsored by the Boehringer Ingelheim Fonds, and the beauty
of the surrounding scenery conferred a peaceful yet spirited environment to
exchange thoughts and ideas.
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Introduction
At this meeting, recent breakthrough findings on the molecular
mechanisms, animal models and, in particular, the therapy of Alzheimer's
disease (AD), and the second most common chronic neurodegenerative disorder
Parkinson's disease (PD), were discussed. Both illnesses are paradigmatic for
an expanding class of late-onset diseases that are characterized by brain
deposits of misfolded proteins. The cross- -sheet conformation of these
pathologically misfolded proteins is a common biophysical feature of
aggregation diseases. Specific dyes (such as thioflavin S) selectively bind to
such so-called amyloid structures, irrespective of the individual protein that
aggregates in each 'amyloidosis'. In the case of AD, the hallmark lesions are
extracellular plaques composed of amyloid- peptides (A) that are
derived from a larger A precursor protein (APP; Fig.
1) and neurofibrillary tangles (NFTs) formed by the
microtubule-associated protein tau. Furthermore, the pre-synaptic protein
 -synuclein (-SYN) fibrillizes into Lewy bodies (LBs), which are
diagnostic for PD but also occur in some dementias including certain variants
of AD. Although the underlying pathogenic cascades and the areas of the brain
most affected are different for each disease, it is becoming increasingly
apparent that the amyloidoses in the brain mutually influence each other, and
experimental approaches used in one field have stimulated research in the
other. Obviously, the amount of information and the broad area of research that
is touched on at meetings such as this cannot all be incorporated into a brief
meeting report. Here, we summarize some of the highlights.
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Figure 1
Cleavage of amyloid- precursor protein. APP can be cleaved by
-secretase (upper, left) or by -secretase (upper right), resulting
in the release of the soluble ectodomains. The APP carboxy-terminal fragments
(C83 and C99, respectively) are substrates for  -secretase. This yields
the p3 or A peptides, which are secreted into the extracellular space,
and the APP intracellular domain fragment C59, which is released into the
cytoplasm. The -secretase is a multiprotein complex and presenilin (PS),
nicastrin, PEN2 and APH1 are required for its full activity (lower part of the
figure). A, amyloid-; APP, A precursor protein; APPs
and APPs, soluble APP, cleaved by - and -secretase,
respectively.
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Intramembrane cleavage
Three years ago, Brown and colleagues defined the concept of
'regulated intramembrane proteolysis' (Brown et al.,
2000), a novel type of intracellular signalling that has been
conserved in prokaryotes and eukaryotes. The two prototypes of this process are
cleavage of the sterol-responsive-element binding protein (SREBP), which
regulates cholesterol metabolism, and Notch, which regulates cell fate.
Cleavage occurs first in the lumenal domain or ectodomain of the protein by a
protease whose activity is regulated. A second cleavage occurs in the
transmembrane domain of the substrate, resulting in the release of the
cytoplasmic domain, which ultimately regulates gene transcription. The
processing of APP that is relevant to the development of AD is initiated by
-secretase followed by intramembrane cleavage mediated by
-secretase (Fig. 1). The latter liberates both a
cytoplasmic domain that might participate in transcriptional control and at the
same time produces the abundant 40-amino-acid soluble A40, as
well as the amyloidogenic 42-amino-acid A42 (Haass & Steiner, 2002). Most familial AD mutations lead
to a pathogenic increase of the A42/A40
ratio, explaining the particular interest in regulated intramembrane
proteolysis at this meeting on neurodegenerative diseases.
The molecular composition of the -secretase complex that is
responsible for the intramembrane proteolysis of APP was one of the hot topics.
In addition to presenilin (PS), which constitutes the catalytic subunit of the
complex, three other proteins—nicastrin, APH1 and PEN2—have been
implicated in the -secretase cleavage process by genetic studies. T.
Iwatsubo (Tokyo, Japan), H. Steiner (Munich, Germany) and D. Selkoe (Boston,
MA, USA) reported that these three proteins, together with PS, form a stable
complex (Edbauer et al., 2003;
Kimberly et al., 2003; Takasugi et al., 2003). When they are expressed
together in vivo, the cleavage rate of APP increases. Downregulation of
any one of the components by RNA interference results in a strong decrease in
-secretase activity, indicating that all four proteins are necessary to
fully constitute activity. In yeast cells that do not have endogenous
-secretase activity, expression of the four proteins together is
sufficient and necessary to give cleavage of APP. The sum of the molecular
weights of the different components is 200–250 kDa, but estimates of this
total based on the electrophoretic motility of the complex in non-denaturing
gels vary between 200 and 550 kDa. Furthermore, considering that APH1 and PS
are both encoded by two different genes—APH1a and APH1b,
and PS1 and PS2, respectively—several combinations of these
proteins can be envisaged (De Strooper, 2003). This
could explain why so many diverse substrates are cleaved by what was, until
recently, considered to be a single -secretase complex. B. Martoglio
(Zurich, Switzerland) broadened the discussion by examining the biology of
signal-peptide peptidase (SPP), which is a presenilin-related protease that
cleaves intramembrane proteins (Weihofen & Martoglio,
2003). SPP generates histocompatibility leukocyte antigen E (HLA-E)
epitopes through the cleavage of signal peptides. Although SPP is a GXGD-type
aspartyl protease like PS (Haass & Steiner,
2002), it is apparently not part of a multiprotein complex. Also,
unlike PS, SPP does not undergo endoproteolysis, and has an opposite membrane
orientation. This is probably the reason why it cleaves type II membrane
proteins, whereas PS cleaves type I proteins. Nevertheless, several inhibitors
that inhibit PS also inhibit SPP, pointing to potential side effects of using
-secretase inhibitors to treat AD.
Therapeutic approaches against extracellular
amyloid
One of the most fascinating and innovative concepts for clearing the
extracellular lesions in the AD brain is immunotherapy. D. Schenk from ELAN
Pharmaceuticals (San Francisco, CA, USA) explained the concept of AD
immunotherapy, which involves the injection of pre-aggregated synthetic A
into patients to elicit an immune response against misfolded A (Dodel et al., 2003). This approach has been validated
in preclinical studies involving transgenic mouse models. ELAN's phase IIa
trial unfortunately had to be stopped because of severe inflammatory side
effects in several patients treated with AN-1792 (the A42
formulation used in the clinical trials). However, the immunized patients are
under further clinical monitoring and the definitive data will be disclosed at
the end of this year. C. Hock (Zurich, Switzerland) provided some positive spin
to the discussion by presenting the preliminary results from the Zurich cohort
of the study. Twenty out of the 30 individuals who participated in this
randomized trial responded to the A immunization by generating antibodies
that recognized the pathological A species but not soluble A or APP
(Hock et al., 2002). In fact, the patients
who developed an immune response against plaques showed a significantly
decreased cognitive decline compared with the rapidly deteriorating control
cohort (Hock et al., 2003). So far, 6% of
the patients have developed post-vaccination complications
(meningoencephalitis), but this grave side effect was treatable with
corticosteroids. A recent case report on one of the patients from the British
cohort of the ELAN study, who developed side effects and later died (Nicoll et al., 2003), suggests that the vaccine has
'clearing' capacities: the temporal cortex of this patient was almost entirely
free of plaques and the other pathologies normally found in their immediate
surroundings, such as plaque-associated dystrophic neurites and astrocytosis.
However, plaque load in other brain regions (such as the frontal cortex)
remained high, and vascular amyloidosis was unaffected even in the brain
sections that were devoid of plaques. NFT pathology was also not suppressed
within the time frame of this patient's immunization. Not surprisingly given
the inflammatory complications, massive lymphocyte invasion into the brain was
persistent even 12 months after the last AN-1792 injection. What was probably
benign recruitment of helper T cells was found in the brain, but an alarming
infiltration of white matter by macrophages was also detected. It remains to be
determined whether or not any benefits gained by immunotherapy outweigh the
risk of potential side effects.
In any event, Schenk suggested dissociating the epitopes in the
A peptide, as the amino-terminal region of A is responsible for the
B-cell response, whereas the middle region generates the T-cell response
(Schenk, 2002). IgG2a subtype
immunoglobulins that recognize N-terminal A epitopes seem to be optimal.
M. Jucker (Basel, Switzerland) pointed out that the cerebral amyloidosis
present in up to 80% of AD patients might be involved in the development of
vascular side effects. In a transgenic mouse model that has a pronounced
cerebral amyloid angiopathy, passive A immunization caused cerebral
haemorrhage (Pfeifer et al., 2002). Thus,
efforts should be undertaken to develop methods to identify patients at risk
with high cerebral amyloid angiopathy scores, possibly by using functional
magnetic resonance imaging.
Another approach to dissolving amyloid plaques is to disrupt their
structure chemically. C. Soto (Plan-les-Ouates, Switzerland) has developed
so-called -sheet breakers, which are peptides that intercalate with the
amyloid-seeding peptide sequence of aggregation-prone proteins and are spiked
with conformation-breaking proline residues. A five-residue
-sheet-breaker peptide (iA5p) specifically prevented A
fibrillization in vitro. Administration of this -sheet breaker
reduced the plaque load and ameliorated astrogliosis and microglial activation
as well as neurodegeneration in a transgenic mouse model. This treatment also
improved water maze performance in a rat model of amyloidosis despite only
partial reduction of plaque burden. Soto reported that the toxicological
effects so far are acceptable and that no immunological responses to
-sheet breaking peptides have been detected. Current optimization
strategies for this approach include the development of small peptidomimetics
and non-hydrolysable peptide derivatives (Adessi et
al., 2003).
Finally, another approach to the treatment of AD would be to prevent
the formation of A and thus inhibit plaque formation altogether. The
three secretases involved in processing APP are obvious targets for
amyloid-modulating approaches (Fig. 1), and
proof-of-concept for - and -secretase inhibition was provided at
the meeting. M. Citron (Thousand Oaks, CA, USA) reviewed the properties of
-site APP-cleaving enzyme 1 (BACE1). The elimination of this
-secretase by knocking out the Bace1 gene in mice abrogated
A formation. More importantly, Citron reported that knocking out the
Bace1 gene in a transgenic mouse model for plaque formation suppressed
pathology, with no adverse effects, due to the elimination of Bace1. Thus,
BACE1 inhibitors should have no side effects, but developing a drug to inhibit
this enzyme may not be straightforward. So far, only peptides have been used to
block the wide and complex active cleft of the BACE1 protease, and the
development of drug-like small-molecule inhibitors of BACE1 remains a challenge
(Citron, 2002).
The complex nature of the membrane-embedded -secretase (see
above) and its many biological functions also make this a challenging drug
target. E.H. Koo (La Jolla, CA, USA) has found that a subset of non-steroidal
anti-inflammatory drugs (NSAIDs) are allosteric inhibitors of -secretase
(Weggen et al., 2001). This effect is
independent of the intended effect of NSAIDs, namely cyclooxygenase inhibition.
The therapeutic value of NSAIDs against AD had been suspected from
retrospective studies of patients who had been prescribed NSAIDs for rheumatoid
arthritis. It was found that the incidence of AD in these patients was
significantly reduced, and the anti-AD potential of NSAIDs was ascribed to the
suppression of the inflammation around plaques. Koo then explained that the
NSAID indomethacin was the only cyclooxygenase inhibitor investigated
clinically for AD that showed efficacy in clinical trials and that inhibited
-secretase. Thus, it may be possible to optimize NSAID derivatives for
maximal -secretase inhibition and minimal cyclooxygenase inhibition.
While the exact molecular mechanism of allosteric -secretase inhibition
awaits elucidation, Koo has begun a prospective, two-centre phase I clinical
trial with R-Flurbiprofen in 48 healthy elderly (55–80-year-old) subjects
to assess its safety, tolerability and pharmacokinetics, as well as blood and
cerebrospinal fluid levels of the biomarker A.
Intracellular amyloid: tau and -synuclein
A model of how the tau protein could interfere with neuronal
viability before fibril maturation was presented by E.-M. Mandelkow (Hamburg,
Germany). The motor protein kinesin moves towards the plus ends of
microtubules, and another motor protein, dynein, moves towards the minus ends.
However, when kinesin slips off the microtubule, tau physically blocks its
reassembly, whereas dynein reassembly is not affected. Thus, dynein-mediated
microtubular transport prevails in the presence of elevated tau levels, and
plus-end transport is inhibited. This leads to the accumulation of axonal
transport cargoes (synaptic vesicles, mitochondria, peroxisomes, and so on) in
the cell soma, which deprives the synapse of vital support and antioxidative
defence systems and, ultimately, causes a 'dying back' of the axon (Stamer et al., 2002). Microtubule affinity
regulating kinases (MARKs) can alleviate this process by phosphorylating tau at
sites that cause its dissociation from microtubules, which de-represses
mitochondrial transport and thereby restores synaptic energy production. It
remains to be shown how the AD-specific phosphorylation of tau influences this
process.
An emerging topic in amyloid research is the reciprocal influence of
the aggregating proteins A, tau and -SYN on each other. E. Masliah
(La Jolla, CA, USA) provided experimental evidence that elevated levels of
A42 enhanced -SYN inclusion body formation in a bigenic
mouse model (Masliah et al., 2001).
Similarly, crossbreeding between plaque mice or the direct injection of A
into tau transgenic mice promoted tau fibrillization (Lee,
2001). It is not yet clear how extracellular A42
impinges on intracellular -SYN and tau. The synergistic fibrillization
of the axonally transported proteins tau and -SYN, as described by
V.M.-Y. Lee (Philadelphia, PA, USA), seems more straightforward, at least from
a spatial point of view. In vitro experiments suggested that all
isoforms of tau and -SYN reciprocally seed each other to form separate
homopolymers (Giasson et al., 2003).
Transgenic mice were engineered to express tau or -SYN in
oligodendrocytes, and amyloid fibrils formed only on crossbreeding the two
lines. Indeed, NFTs and LBs were occasionally observed in the same neuron.
J.Q. Trojanowski (Philadelphia, PA, USA) and P.J. Kahle (Munich,
Germany) reported on the recent achievements in recapitulating LB pathology in
transgenic mouse models that express human mutant -SYN (Giasson et al., 2002; Neumann
et al., 2002). In an age- and gene-dose-dependent manner,
these animals developed fibrillar -SYN deposits within neurites and
neuronal perikarya and showed all of the traits of human pathology that are
concomitant with lethal locomotor deterioration. Remarkably, the dopaminergic
neurons in the midbrain, the degeneration of which accounts for parkinsonian
symptoms in human patients, were consistently unaffected in the transgenic
mouse models. By contrast, D. Kirik (Lund, Sweden) and P. Aebischer (Lausanne,
Switzerland) reported that viral delivery of high gene doses of -SYN
into the substantia nigra did result in dopaminergic neurodegeneration
(Kirik et al., 2002; Lo
Bianco et al., 2002). This illustrates that a combination of
transgenic technology and viral gene transfer considerably expands our
experimental toolkit for the study of neurological disease. Aebischer went on
to give an overview of the potential of lentiviral gene transfer to generate
animal models and therapeutic approaches for neurodegenerative diseases. One
approach would be to downregulate dominant genes or pathologically active
enzymes (such as APP, PS, BACE1 and -SYN) through the introduction of
small interfering RNAs using lentiviral vectors. A second approach would be to
restore the expression of recessive genes (such as parkin and DJ1).
Third, neuroprotective and neurotrophic genes could be delivered to affected
tissue. It might even become possible to optimize the therapeutic doses for
individual patients by the use of tetracycline-regulatable lentiviral
constructs.
Conclusions and perspectives
This was an exciting meeting that showed the enormous research
efforts in the fields related to amyloid diseases. It is remarkable that
amyloidogenic proteins such as tau, -SYN and A mutually influence
each other's aggregation properties, which leaves one wondering whether common
molecular mechanisms or common molecular principles underlie the two major
neurodegenerative disorders AD and PD. At least, the fact that so many
researchers cross the borders between these two pathological entities is
remarkable. A great deal has been learned in recent years about the
pathogenesis of both disorders, and a general feeling of optimism spread
throughout the meeting when looking at the spectacular results obtained with
A vaccination in AD patients or with viral vectors that express growth
factors in PD models. We look forward to future meetings focusing on drugs and
therapy, and to evaluating the clinical progress in these fields.
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Acknowledgements
This meeting was sponsored by the Boehringer Ingelheim Fonds
(Foundation for Basic Research in Medicine). We apologize to the participants
whose work could not be cited here due to space limitations.
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