Beta Amyloid Definition
Amyloid beta (Aβ or Abeta) is a peptide of 39–43 amino acids that appear to be the main constituent of amyloid plaques in the brains of Alzheimer's disease patients. Read More
Research Overview
Accumulation
of b-amyloid
protein (Ab)
in the brain causes changes in neuritic processes in individuals with
this
disease. Steven Petratos et al., 2008 (1) have showed that Ab
decreases neurite outgrowth from SH-SY5Y human neuroblastoma cells. To
explore
molecular pathways by which Ab
alters
neurite outgrowth, we examined the activation
and localization of RhoA and Rac1 which regulate the level and
phosphorylation
of the collapsin response mediator protein-2 (CRMP-2). Ab
increased the levels of the GTP-bound (active) form of RhoA in
SH-SY5Ycells.This increase in GTP-RhoA correlatedwith an increase in an
alternatively spliced formof CRMP-2 (CRMP-2A) and its threonine
phosphorylated form.
Both a constitutively active form of Rac1 (CA-Rac1) and the Rho kinase
inhibitor, Y27632, decreased levels of the CRMP-2A variant and
decreased
threonine phosphorylation caused by Ab
stimulation. The amount of tubulin bound to CRMP-2
was decreased in the presence of Ab
but Y27632
increased the levels of tubulin bound to
CRMP-2. Increased levels of both RhoA and CRMP-2 were found in neurons
surrounding amyloid plaques in the cerebral cortex of the APP(Swe)
Tg2576
mice.We found that there was an increase in threonine phosphorylation
of CRMP-2
inTg2576 mice and the increase correlated with a decrease in the
ability of
CRMP-2 to bind tubulin.The results suggest that Ab-induced
neurite outgrowth inhibition may be initiated through a mechanism in
which Ab
causes an increase in Rho GTPase activity which, in turn,
phosphorylates CRMP-2
to interfere with tubulin assembly in neurites.
In
another
research theory reduced brain insulin signalling
and low CSF-to-plasma insulin ratios have been observed in patients
with
Alzheimer disease (AD). Furthermore, intracerebroventricular or IV
insulin administration
improve memory, alter evoked potentials, and modulate
neurotransmitters,
possibly by augmenting low brain levels. After intranasal
administration,
insulin-like peptides follow extracellular pathways to the brain within
15
minutes.
Beta
amyloid and Alzheimer’s disease:
The
b-amyloid
protein of Alzheimer’s
disease increases neuronal CRMP-2 phosphorylation by a Rho-GTP
mechanism:
The
generation of Ab
and the deposition of amyloid is associated with neuronal dysfunction
and loss
of functional synapses caused by changes to neurite morphology (2
&3).
The neuritic changes in neurons of the frontal and temporal cortices
may
initially lead to a mild cognitive impairment (MCI), that is followed
by more
severe memory loss as the disease progresses (4). There is strong
evidence for
the involvement of extracellular Ab
in the disruption of the integrated neuronal circuitry (5) It has been
examined
the effects of Ab
on neurite outgrowth and to determine the activation of downstream
signalling
mechanisms which mediate these effects. In human neuroblastoma SH-SY5Y
cells, Ab
can reduce
the length of neuritis by inactivating
the neurite outgrowth signalling molecule Rac1, and that this Ab-mediated
reduction in neurite length can be
abrogated by the Rho Kinase inhibitor, Y27632. Furthermore, researchers
have
showed that the Ab-mediated
decrease in neurite length involves the induction of a threonine
phosphorylation of CRMP-2A, conferring a reduced binding capacity to
tubulin,
both of which can be reversed by inhibiting RhoA activity. Importantly,
evidence
validated that this mechanism also operates in the Tg2576 mouse model
of AD,
where RhoA and CRMP-2 are increased in the immediate vicinity of
amyloid plaques
and CRMP-2-bound tubulin is reduced. The data suggest that Ab-mediated
neurite outgrowth inhibition is initiated
through the activity of RhoA-GTP and through the dysregulation of
CRMP-2 to
bind tubulin for neurite outgrowth.
As
Ab
deposition is associated with neuritic abnormalities in the AD brain
(6),
authors investigated the effect of different Ab
peptides on neurite outgrowth. Freshly added or
‘aged’
(aggregated) Ab40
and Ab42
(1 mM) were added to RA-differentiated SH-SY5Y cells. Ab40
caused a
significant reduction in neurite length
over a 24-h incubation period (Fig. 1). Ab40
induced an approximate 40% reduction in neurite length when added fresh
(not
aged) (Fig. 1A and C). When Ab40
that had been incubated for 7 days at 37C (aged) was used, a reduction
in
neurite length of ~25%
was observed (Fig. 1B and D). There was no significant reduction in
neurite length
of SH-SY5Y cells incubated with Ab40
or Ab42
peptides containing a scrambled sequence of amino acids (scrambled Ab40
and Ab42)
(Fig. 1A and B). Fresh Ab42
(1 mM) also produced a reduction in neurite length of ~25%
(Fig. 1A). Surprisingly, after ageing Ab42,
there was a decrease in the level of neurite outgrowth inhibition with
only a
10–15% reduction in neurite length (Fig. 1B). It was
speculated
that because
the Ab
peptides were incubated in the medium for 24 h before the neurite
outgrowth
effects were observed, it was likely that the peptides had already
aggregated
to some extent and further ‘ageing’ of Ab42
may have produced high molecular weight aggregates that were no longer
toxic.
Fig.1
Effect
of fresh and aged Ab on neurite length in
differentiated human SH-SY5Yneuroblastoma cell cultures. Ab
peptides (1
mM) where freshly prepared (A,C) or aged
for 7 days inDMEM/F12 and10% FCS at 37C (B,D). Peptides were added to
cells for
24 h. Panels A and B show quantitation of neurite outgrowth. Panels C
and D are
representative photomicrographs of SH-SY5Ycells incubated with Ab40
and Ab42
peptides (bar=50 mm). (A, _#+P<0.001,
^P<0.05; B, _#^ P<0.001).
Role
of the
familial Dutch
mutation E22Q in the folding and aggregation of the 15–28
fragment of the
Alzheimer amyloid-b
protein
The
presence
of amyloid fibrils in the brain is a clinical hallmark of
Alzheimer‘s disease
(AD). The fibrils consist of large ordered aggregates of amyloid-b
(Ab)
peptides, proteolytic by-products of the enzymatic cleavage of the
Alzheimer
amyloid precursor protein (APP). AD can be sporadic (occurring in
elderly
patients and characterized by a slow progression) or familial (a
hereditary
form characterized by early onset of the disease and aggravated
severity). The
majority of familial forms of AD involve single-point mutations in the
22–23
segment of the Abpeptide.
The focus of this work is on the Dutch form of AD, which involves a
mutation
encoded by a point substitution G to C at codon 693 of the amyloid
precursor
protein (APP). The result is the production of an Ab
peptide
in
which residue E22 is mutated to Q (E22Q mutation). Patients who have
this mutation
are at risk of developing cerebral amyloid angiopathy that typically
leads to
cerebral hemorrhage and stroke. Many in
vitro experiments show
that the AbE22Q
peptide
aggregates much more readily than its
wild-type (WT) counterpart (7-9). As a free monomer in solution, the WT
Abpeptide
is
for the most part unstructured with
residual structure observed locally in a few regions of the primary
sequence (10).
The Ab
peptide can populate a variety of oligomeric species, some of which may
be toxic
(such as transmembrane pores) (11), on route to the fibrillar state.
Once bound
to the fibril, the peptide adopts a b-sheet
conformation (12), implying that fibrillization is accompanied by a
major
structural reorganization. In the case of the E22Q mutation,
experiments by
Maggio et al. (13)
indicate that the rate of Ab
monomer
deposition onto fibrils is enhanced compared
with the WT. The refolding of monomeric Ab
into a conformation commensurate with the fibril presents a free energy
barrier
to fibril growth. Researchers are aiming of the twofold work: (i)
to
investigate how the E22Q mutation affects monomer Ab
conformations and (ii)
to investigate how the E22Q mutation
affects the
deposition reaction of monomers onto fibrils. In aim i,
the
focus on the
effect of the E22Q mutation on two critical regions implicated in Ab
monomer
folding and aggregation, the E22–K28 region
and the L17–A21 central hydrophobic cluster (CHC). NMR and
molecular dynamics
simulations have shown that the 22–28 region adopts a bend
structure in the context
of the proteolytically resistant 21–30 fragment of A Ab
(14–17) and is likely the most structured region of
the full-length Ab40
and Ab42
peptides (14). Further simulations in our group found this same bend
region in
fragments Ab12–28
(18) and Ab10–35
(19). The CHC, on the other hand, appears to serve a s a recognition
site for
monomer binding to the fibril. Mutations in this segment (such as F19T)
completely
abrogate fibril growth (20). It was considered that two model peptides:
fragments 21–30 and 15–28 of the Ab
peptide. The former will enable us to study the effect of the E22Q
mutation on
the structured bend motif while the latter will let researchers probe
the
effects of this mutation on the structure of the CHC region. Although
we are
not considering fragments that include residues distant in sequence
space from residue
22, nonetheless expect to capture the essential effect of the E22Q
mutation.
Indeed, experiments on Ab12–28
(21) and Ab10–35
(7) have shown that the E22Q mutation only induces local changes to the
folding
properties of the peptide that do not extend beyond neighboring
residues of
E22. The simulations involved explicit solvent and the replica exchange
protocol to achieve a thorough sampling of conformational space. In aim
ii,
investigated how the E22Q mutation affects deposition rates onto
preexisting
amyloid fibrils by identifying the transition state (TS) ensemble for
the
fibril elongation reaction and calculating the activation free energy
for
monomer deposition onto preexisting fibrils for the WT and mutant
cases.
References:
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Petratos,
S., Li, Q-X., Amee, J., Xu Hou, G., Kerr,
M. L.. Unabia, S. E., Hatzinisiriou, I., Marie-Isabel, D. M., and
Small, D. H.
2008. The b-amyloid
protein of Alzheimer’s disease increases neuronal CRMP-2
phosphorylation by a
Rho-GTP mechanism. Brain 131, 90-108.
2.
Tsai, J.,
Grutzendler, J., Duff, K., Gan, W.B. 2004.
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Spires,
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Knowles,
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JP, Mcvoy L, Van Nostrand WE (2000) Charge
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K, et al. (2003)
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mutant
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the mechanism
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