neur3904 the potential roles of ampa receptor dysfunction in alzheimer's disease
TRANSCRIPT
The potential roles of AMPA receptor dysfunction in Alzheimer’s disease
Author: Georgios LouloudisSUPERVISOR: DR IAN COOMBS
Number of text pages: 35
Number of figures: 6
Number of tables: 0
Total word count: 7492
Word count of Abstract: 272
Word count of Introduction: 599
Word count of Discussion: 849
Number of References: 72
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Contents
Abstract.......................................................................................................................................2
Declaration of contribution.........................................................................................................2
Introduction.................................................................................................................................3
Ca2+/calmodulin-dependent protein kinase II.............................................................................5
Figure 1........................................................................................................................6
Calcineurin..................................................................................................................................8
Figure 2......................................................................................................................10
Figure 3......................................................................................................................11
Protein Kinases A and C...........................................................................................................13
Figure 4......................................................................................................................15
Mitochondrial dysfunction and caspases..................................................................................17
Figure 5......................................................................................................................20
APOE ε4, TARPs and phospholipid pathways.........................................................................22
Figure 6......................................................................................................................23
Discussion…………………………………………………………………………………… 23
Acknowledgements...................................................................................................................26
Bibliography.............................................................................................................................26
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Abstract
Synaptic dysfunction is a critical neuropathological feature of Alzheimer’s disease (AD). It
may result from the disturbance of physiological long-term potentiation (LTP) and depression
(LTD). In these two synaptic events, AMPA receptors (AMPARs) are normally either
phosphorylated or dephosphorylated and are inserted into the synaptic membrane of neurons
or are internalised. Activation of ionotropic glutamate receptors (iGluRs) in AD has also been
implicated in Ca2+ dysregulation and excitotoxicity. The main aim of this dissertation was to
understand how AMPAR dysfunction occurs in AD and how it contributes to AD-related
neurotoxicity. It was determined that amyloid-beta (Aβ) reduces the activity of
Ca2+/calmodulin-dependent protein kinase II (CaMKII), which is then linked to decreased
phosphorylation of S831-GluA1 and decreased AMPAR presence at the synaptic membrane,
while membrane GluA2 levels remain unaltered. Aβ and tau dephosphorylate S845-GluA1
via calcineurin (CaN), resulting in GluA1 removal from the synaptic membrane. Protein
kinase C (PKC)-mediated phosphorylation of S880-GluA2 at the synapse drives membrane
GluA2 downregulation, whereas protein kinase A (PKA)-mediated phosphorylation of S845-
GluA1 facilitates membrane insertion of Ca2+-permeable AMPARs (CP-AMPARs) without
any effect on surface GluA2/3. Both PKA- and PKC-mediated mechanisms of AMPAR
dysfunction contribute to intracellular Ca2+ dysregulation, an effect that also occurs when Aβ
activates NMDA receptors (NMDARs) and AMPARs. Ca2+ dysregulation then leads to
mitochondrial dysfunction, caspase activation and membrane AMPAR downregulation.
Additionally, decreased RNA editing of GluA2 has been observed in AD patients that are
carriers of APOE ε4, the major genetic risk factor for AD. The roles of AMPAR dysfunction
in AD remain controversial. Further understanding of AMPAR postsynaptic plasticity could
assist in the identification of additional roles of AMPAR dysfunction in AD.
Declaration of contribution
The author declares that this dissertation is his own work.
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Introduction
Alzheimer’s disease (AD) is the most prevalent and severe disorder of human intellect
(Hardy and Selkoe, 2002). It is characterized by progressive dementia, amnesia and cognitive
dysfunction, along with pathological features in the brain, such as intraneuronal
neurofibrillary tangles (NFTs), extracellular amyloid-beta (Aβ) deposits, and synaptic loss
(Dubois et al., 2010). Statistical data obtained from AD patients have shown that cognitive
impairment correlates better with synaptic deficits than with amyloid plaques and
neurofibrillary tangles (Terry et al., 1991). Additionally, it is widely accepted that amyloid
neurotoxicity mediates synaptic abnormalities in AD (Lacor et al., 2004; Shankar and Walsh,
2009; Mucke and Selkoe, 2012).
AMPA receptors (AMPARs) are ionotropic glutamate receptors (iGluRs) that facilitate fast
excitatory synaptic neurotransmission. The assembly of at least two of the four AMPAR
subtypes, GluA1-GluA4, results in the formation of heterotetrameric ligand-gated ion
channels (Nakagawa, 2010). Glutamate binding to the extracellular surface activates the
channels, allowing Na+ and K+ to permeate through, and eliciting excitatory postsynaptic
currents (EPSCs). Editing of the GluA2 RNA by ADAR2 converts a glutamine codon into an
arginine codon, rendering most AMPARs impermeable to Ca2+ (Wright and Vissel, 2012). C-
terminal PDZ-binding domains on AMPARs bind proteins with PDZ domains (Kim et al.,
2001). One such protein is postsynaptic density 95 (PSD-95), which incorporates AMPARs at
synapses in an activity-dependent manner (Ehrlich and Malinow, 2004). Additionally,
transmembrane AMPA regulatory proteins (TARPs) e.g. Stargazin, facilitate the interaction
between AMPARs and PSD-95 (Bats et al., 2007). Given their elaborate structure and
signalling events they participate in, AMPARs are likely involved in AD via highly diverse
ways. AMPAR reduction in regions most affected by AD, e.g. entorhinal cortex and CA1
region of hippocampus, reportedly occurs prior to NFT formation (Ikonomovic et al., 1997;
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Zhao et al., 2010) and, therefore, AMPAR deficits could be occurring early in AD (Zhao et
al., 2010).
Long-term potentiation (LTP) and long-term depression (LTD) are neurophysiological,
synaptic events that underlie cognition. In LTP, transient, high-frequency stimulation (HFS)
of synapses induces a persistent increase in the strength of synaptic connections (Lisman et
al., 2012). Initially, the activation of a sufficient amount of AMPARs drives the rapid
depolarisation of postsynaptic membranes, and the subsequent activation of NMDA receptors
(NMDARs), whose Mg2+ inhibition is removed at depolarising potentials (Fleming and
England, 2010). High Ca2+ conductivity through opened NMDARs causes the activation of
Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Lisman et al., 2012). CaMKII then
drives more AMPARs to synapses by phosphorylating the GluA1 AMPAR subtypes at S831
(Hayashi, 2000). Protein kinase C (PKC) also phosphorylates GluA1 at S816 and S818,
driving the insertion of AMPARs into synapses (Lin et al., 2009). On the contrary, LTD acts
to decrease the strength of synaptic transmission (Bliss and Cooke, 2011). Upon low-
frequency stimulation (LFS), weak calcium signals favourably activate calcineurin (CaN), a
phosphatase that dephosphorylates S845 of GluA1 (Li et al., 2012). S845-GluA1
dephosphorylation is associated with AMPAR endocytosis (He et al., 2011). Overall, the net
effect of LTP and LTD is the insertion and removal, respectively, of AMPARs into synaptic
membranes.
In the present dissertation, the potential ways by which AMPAR function is impaired in AD,
as well as the relationship between AMPAR dysfunction and AD-related neurotoxicity, will
be discussed. This will be achieved by focusing on various signalling events and their link to
AMPAR function, e.g. AMPAR endocytosis and trafficking, caspase activation, etc. The
ultimate objective is to establish the potential roles and importance of AMPAR dysfunction in
AD, and in doing so, conclude whether it acts as a mere biomarker for the disease or whether
it presents opportunities for the development of effective treatments.
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Ca2+/calmodulin-dependent protein kinase II
It is very probable that Aβ species contribute to the signalling deficits in AD by diminishing
the activation of signalling components involved in LTP and synaptic strengthening. Zhao et
al. (2004) claimed that Aβ inhibits the autophosphorylation of CaMKII during LTP. Early
LTP was induced in single rat hippocampal slices as they underwent HFS (100 Hz for 1s) in
the presence or absence of Aβ. Electrophysiological recordings were derived from the dentate
gyrus. Protein lysates were prepared from dentate slices and were probed for phosphorylated
and unphosphorylated forms of GluA1 and CaMKII by immunoblotting. It should be noted
that S831 on GluA1 is a CaMKII phosphorylation site (Barria et al., 1997), whereas S845-
GluA1 is a Protein Kinase A (PKA) site (Banke et al., 2000). The researchers observed an
increase in fEPSP, αCaMKII phosphorylation and GluA1 phosphorylation at S831, when LTP
was measured in the absence of Aβ. However, when Aβ was applied, dentate LTP was
inhibited, CaMKII phosphorylation and GluA1 phosphorylation at S831 were reduced, but
phosphorylation S845-GluA1 was unaffected. The levels of total GluA1 did not change
regardless of the presence of Aβ.
Reduced CaMKII activity by Aβ and the subsequent diminution of GluA1 phosphorylation
could potentially lead to impaired AMPAR trafficking and reduced AMPAR current flow.
This is at least partly due to phosphorylation of TARPs, which has been shown to activate the
diffusional entrapment of membrane AMPARs (Opazo et al., 2010). Gu et al. (2009)
measured the levels and distribution of CaMKII, p-CaMKII, GluA1, GluA2, pS831-GluA1,
pS880-GluA2 and GluN1 in APPSwe transgenic mice that overexpress AD-related mutant
APP, and age-matched wild-type (WT) mice, by preparing subcellular fractions from the
frontal cortex. It was revealed that the total levels of all the proteins of interest differed
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negligibly between APP and WT mice (see figs. 1A, 1B). However, their distribution differed
as the levels of both CaMKII and pCaMKII were significantly higher in the cytosolic fraction
of APP mice compared to that of WT mice, whereas the levels of membrane-associated
CaMKII and p-CaMKII declined in the APP mice relative to WT mice (see fig. 1A). As there
is decreased availability of CaMKII and p-CaMKII at the membranes of frontal cortex
neurons, these findings raise the expectation that phosphorylation of GluA-S831 will be
reduced in APP mice, potentially resulting in downregulation of surface GluA1. Indeed, the
levels of pS831-GluA1 and membrane-associated GluA1 were significantly lower in APP
mice (see fig. 1B). AMPAR-EPSCs were recorded in Aβ-treated cortical pyramidal neurons.
AMPAR-EPSCs were significantly diminished in the APP transgenic mice compared to the
WT mice, whereas such an effect was not observed with NMDAR-EPSCs (see figs. 1C, D).
Gu et al. (2009) finally observed that knocking down CaMKII imitated the Aβ-mediated
reduction of AMPAR currents in cortical neurons, whereas overexpression of CaMKII
increased the amplitude of AMPAR currents. NMDAR currents were insignificantly affected
by either experimental manipulation (see fig. 1E, 1F).
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Fig. 1 Aβ mediates the translocation of CaMKII and the synaptic removal of GluA1. Described according
to information from Gu et al. (2009). Proteins were subcellularly fractionated. S, the cytosolic fraction; P1, the
synaptosomal fraction that corresponds to the cytosol of the synaptosome; P2, the synaptosomal fraction that
corresponds to the synaptic membrane. Protein levels were quantified by Western Blotting. A. Percentage
change of CaMKII-α, CaMKII-β and p-CaMKII in the subcellular fractions of APP mice relative to WT mice.
Mean ± SE; *, p<0.01, ANOVA. B. Percentage change of total and surface GluA1, pS831-GluA1, total GluA2,
pS880-GluA2, total and surface GluN1 in APP mice compared to WT mice. Mean ± SE; *, p<0.01, ANOVA.
AMPAR-EPSCs and NMDAR-EPSCs were recorded by whole-cell patch clamp in pyramidal neurons of cortical
slices from APP and WT mice. AMPAR-EPSCs were elicited by stimulation pulses (5.5 V, 0.05 ms) and
recorded from neurons at -70 mV. NMDAR-EPSCs were evoked by stimulation pulses (6.5 V, 0.5 ms), after
neurons were depolarised from -70 mV to +60 mV for 3 seconds. C. Measurement of AMPAR- and NMDAR-
EPSCs in 3- and 6-month-old APP and WT mice. Scale bar, 50 pA, 50 ms (AMPAR-EPSCs) or 200 ms
(NMDAR-EPSCs). D. Quantification of the amplitude of AMPAR- and NMDAR-EPSCs and the AMPAR-
EPSC/NMDAR-EPSC ratio in 3- and 6-month-old APP and WT mice. Mean ± SE; *, p<0.01, ANOVA. E.
Whole-cell AMPAR and NMDAR currents of cortical neurons were measured after the neurons were either
transfected with CaMKII siRNA or CaMKII, and treated with either vehicle or 0.1 mM Aβ1-42 solution. F.
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Quantification of AMPAR and NMDAR currents in cortical neurons transfected with either GFP, CaMKII
siRNA and CaMKII, and either treated or not treated with Aβ. Mean ± SE; *, p<0.01, ANOVA. Adapted from
Gu et al., 2009.
Both papers strongly support that Aβ acts to reduce the interaction between CaMKII and
AMPARs, which is important for enhanced synaptic strength. However, Zhao et al. (2004)
argues that Aβ may be inhibiting the autophosphorylation of CaMKII, whereas Gu et al.
(2009) claim that Aβ causes CaMKII mislocalisation and the subsequent synaptic removal of
GluA1. Zhao et al. (2004) could have also considered measuring the levels of synaptic
GluA1. They showed that Aβ resulted in impaired hippocampal LTP via electrophysiological
recordings of the performant pathway, but they did not specify whether that impairment was
due to impaired AMPAR activation or due to the lower AMPAR surface numbers. On the
other hand, the strong point of the study by Gu et al. (2009) is that it shows that the Aβ-
induced reduction in AMPARs is correlated with a reduction in AMPAR-evoked current
amplitudes. Even more important was the fact that they carried out electrophysiological
recordings of NMDARs and measured synaptic NMDAR levels. An assumption that was
made by Zhao et al. (2004) is that the reduced CaMKII activity is brought about by the
inhibition of NMDAR-evoked currents and their study could have been strengthened if they
tested that assumption. Gu et al. (2009) showed that neither the levels nor the currents of
NMDARs are significantly affected, implying that AMPARs may be playing a much more
important role in AD via their decreased presence at the synaptic membrane.
Calcineurin
CaN is also important in AD-related synaptic dysfunction. The CaN inhibitor FK506 has
been observed to reverse LTD and the reduction of dendritic spine density in organotypic
hippocampal slices, two effects that are facilitated by Αβ (Reese and Taglialatela, 2011).
Chen et al. (2002) performed a similar experiment. They observed that bath application of 8
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0.2-1.0 μM Aβ during a 3h-long late-phase LTP (L-LTP) that was induced by HFS trains in
the medial performant path of rat (25-45-day-old Sprague-Dawley) hippocampal slices, halted
both initial and late stages of L-LTP, with the late stage being primarily affected during the
first hour of L-LTP. Application of FK506 completely blocked the L-LTP deficits that were
induced by Aβ. However, Aβ weakened NMDAR-mediated EPSCs without affecting
AMPAR-mediated EPSCs.
In another study, Zhao et al. (2010) mostly used biotinylated Aβ-derived diffusible ligands
(bADDLs). The bADDLs were found to rapidly bind dendritic spines and colocalise with
spinophilin, a synaptic marker for the detection of dendritic spines involved in excitatory
neurotransmission. 60 minutes after murine neuroblastoma N2A cells were treated with
bADDLs, most bADDLs were internalised and brought to the cytosolic compartment.
Knockdown of the catalytic domain of CaN with Pppca3 siRNA resulted in attenuation of
bADDL internalisation. A similar effect was observed when the researchers knocked-down
the genes for AMPAR subtypes, and by immunocytochemistry they revealed that bADDLs
exhibited greater degrees of binding to dendrites bearing abundant dendritic spines and
GluA2, in primary hippocampal neurons from 18-month-old Sprague-Dawley rats. The
researchers then employed immunofluorescent colocalisation and acid stripping experiments
to show that bADDLs, endogenous Aβ and AMPARs are trafficked to endosomes, an effect
which was blocked by the CaN inhibitor FK506. By photoreactive amino acid cross-linking,
Zhao et al. (2010) supported that endogenous Aβ interacts with GluA2/3 or with proteins that
are complexed to those receptors. Finally, Zhao et al. (2010) observed that CNQX, IEM1064
and GYKI52466 managed to inhibit bADDL-AMPAR binding, but only CNQX, the
competitive AMPAR inhibitor, and GYKI52466, the negative allosteric modulator, prevented
AMPAR loss. As Ca2+-permeable AMPARs (CP-AMPARs) lack the Ca2+-impermeable
GluA2 subunit and IEM1064, the CP-AMPAR inhibitor, failed to halt bADDL-induced
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AMPAR loss, the Ca2+-impermeable GluA2 subunit might be the primary target for these
internalisation events (Zhao et al., 2010).
Miller et al. (2014) claim that tau and CaN are the signalling components that mediate Aβ-
induced AMPAR signalling deficits. Tau may have a critical physiological role at the synapse,
as AMPAR internalisation can be reduced in tau KO mice and, additionally, tau
phosphorylation at S396 can contribute to hippocampal LTD via a potential enhancement of
GluA2-PICK1 association, which acts to drive GluA2 endocytosis (Regan et al., 2015).
Firstly, Miller et al. (2014) observed that the proportion of GFP-tagged WT tau that
mislocalised to the dendritic spines in vitro was greater in APPSwe mice than transgenic-
negative (TgNg) mice. The researchers also observed that the amount of tau in dendritic
spines of cultured hippocampal neurons increased upon treatment with 2 μM oligomerised
Aβ1-42 solution. The latter effect was abolished in cells that were transfected with AP tau, a
phosphorylation-resistant tau mutant (see figs. 2A, 2B). By measuring AMPAR-mediated
miniature EPSCs (mEPSCs), Miller et al. (2014) revealed that WT tau-transfected neurons
exhibited decreased AMPAR-mediated mEPSC amplitudes when treated with Aβ, an effect
which was abolished in AP tau-transfected neurons (see figs. 2C, 2D). Therefore, Aβ-
mediated disruption of AMPAR function seems to require the presence of phosphorylated tau
in dendritic spines.
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Fig. 2 Tau mediates Aβ-induced synaptic and AMPAR deficits. Described according to information from
Miller et al. (2014). A. In vitro images of cultured hippocampal neurons, transfected with GFP-AP tau and
DsRed, untreated (upper panel) or treated (middle and bottom panels) with Aβ1-42. Images of neurons one day
(middle panel) and three days (lower panel) after Aβ1-42 treatment. Arrowheads indicate spines lacking tau. All
images were processed with MetaMorph. Scale bar, 10 μm. B. Quantification of % spines containing tau as the
number of tau-containing spines was divided by the total spine count. Spines were counted manually. The
percentage of spines containing tau did not change significantly after treatment with either vehicle or Aβ1-42
solution. Repeated-measures two-way ANOVA, Bonferroni post-test. C. AMPAR-mEPSCs were measured in
cultured dissociated rat hippocampal neurons, 21-25 days after Aβ treatment, at -55 mV. AMPAR-mEPSCs were
measured under the effect of WT Tau or AP Tau and vehicle or Aβ1-42 solution. D. Mean ± SEM AMPAR-
mEPSC amplitude from WT Tau and AP Tau neurons treated with Aβ or vehicle solution. Two way ANOVA,
Bonferroni post-test, **P<0.01. Adapted from Miller et al., 2014.
In the final parts of their experiment, Miller et al. (2014) observed that treatment with FK506
would yield similar AMPAR-mEPSCs in control-treated and Aβ-treated neurons (see figs.
3A, 3B). Using GluA1 S845A mice, whose GluA1 subunit cannot be phosphorylated at S845,
and the anti-N-GluA1 antibody that recognises surface AMPARs, they observed that Aβ
treatment reduced GluA1 numbers on dendritic spines of neurons with WT GluA1, whereas 11
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that observation was not made for neurons bearing GluA1 S845A. Finally, the researchers
reported that expression of tau in the P301L tauopathy mouse model diminished membrane
AMPAR numbers on dendritic spines, an effect that was rescued by the application of FK506
(see figs. 3C, 3D). Therefore, Miller et al. (2014) concluded that both tau and CaN are
important for Aβ-mediated AMPAR signalling deficits.
Fig. 3 The CaN inhibitor FK506 rescues AMPAR signalling deficits. Described according to information
from Miller et al. (2014). A. Hippocampal neurons were either treated with vehicle, Aβ1-42, vehicle + FK506, or
Aβ1-42 + FK506 solution at 19-22 days in vitro (DIV) and AMPAR-mEPSCs were recorded three days after
treatment. B. Quantification of the amplitude of AMPAR-mEPSCs from Aβ1-42-treated and vehicle-treated
neurons in the presence and absence of FK506. Mean ± SEM. Two-way ANOVA, Bonferroni post-test,
*P<0.05, **P<0.01. C. Representative images of cultured hippocampal neurons from negative transgenic (TgNg)
mice that do not recapitulate AD, and P301L Tau transgenic mice. The neurons were treated with anti-N-GluA1
(green) and anti-PSD-95 (red) antibodies. Specifically, the anti-N-GluA1 antibody recognises the N-terminus of
GluA1. At 14-16 DIV, the cells were incubated with FK506 or control solution and the images were taken at 21
DIV. The arrows pinpoint the GluA1 clusters that co-localised with PSD-95 clusters; the arrowheads indicate
GluA1 in dendritic shafts. All images were analysed with MetaMorph. Scale bar, 10 μm. D. Quantification of N-
GluA1 signal of vehicle-treated TgNg neurons, Aβ1-42-treated TgNg neurons, vehicle-treated P301L Tau neurons
and Aβ1-42-treated P301L Tau. The signal was assessed at single dendritic spines and adjacent dendritic shafts.
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Two-way ANOVA, Bonferroni post-test, n=10 neurons per group, ***P<0.001. Adapted from Miller et al.,
2014.
Aβ is considered to initiate or accelerate tau pathology early in AD and, as the disease
progresses, an Aβ deposition threshold is reached, after which tau acts as an independent
disease-conferring agent (Lansdall, 2014). The study by Miller et al. (2014) seems to be in
line with that speculation, as AMPAR-signalling deficits may be occurring early in the
disease (Zhao et al., 2010) and Aβ treatment increased tau insertion into spines. Miller et al.
(2014) could also have revealed the ages of the mice used for their study, to offer insight
regarding the stage and the timescale at which these signalling events take place in AD.
However, it should also be considered that both APPSwe and P301L are single mutants of APP
and tau, respectively, that do not robustly display the other type of pathology (Kitazawa et al.,
2012) and thus do not accurately recapitulate AD. The researchers concluded that the CaN
inhibitor FK506 reduces the effects of both Aβ treatment and pathological tau on AMPAR
numbers, but they focused on each pathological feature separately. They could have looked
into the effect of FK506 on P301L hippocampal neurons in the presence or absence of
endogenous Aβ or they could have used a mouse model that better recapitulates AD, e.g. the
triple transgenic APP/PS1/htau mouse model (Guo et al., 2013). Additionally, GluA1 S845A
is resistant to either phosphorylation or dephosphorylation at S845 (Miller et al., 2014). Their
GluA1 receptors should primarily be unphosphorylated at S845A and it is thus difficult to
assess the effect of CaN, a phosphatase that dephosphorylates S845-GluA1 (Li et al., 2012).
At the same time, other kinases would not have been able to phosphorylate S845-GluA1, e.g.
cyclic guanosine monophosphate-dependent protein kinase II (Xue et al., 2014), and these
could have also been held accountable for AMPAR signalling deficits. It was recently shown
that treatment of cultured hippocampal neurons from E17-18 PS1 M146V mice, i.e. mice that
recapitulate early-onset AD by expressing mutant presenilin 1 (PS1), with 5 μM FK506,
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increased S845 phosphorylation and elevated surface GluA1 levels (Kim et al., 2015), so this
phosphorylation site could potentially prove useful as a potential AD therapeutic target.
The overall picture that arises from the studies of Zhao et al. (2010) and Miller et al. (2014)
is that AMPARs, especially GluA2, are important for the postsynaptic membrane localisation
of soluble Aβ species, which mediate tau hyperphosphorylation. Tau then favours CaN
activity, which in turn dephosphorylates S845-GluA1 and drives the endocytosis of
AMPARs, ultimately contributing to LTP impairments and synaptic deficits in AD. How all
these processes occur though remains to be understood and it has even been suggested that
CaN actually mediates tau hyperphosphorylation via GSK-3β (Tu et al., 2014). What is also a
bit troubling is that Chen et al. (2002) confirm that FK506 treatment reverses Aβ-induced
LTP deficits, but argue that AMPARs are not affected by Aβ. However, it should be
considered that Chen et al. (2002) looked into L-LTP. Co-expression of APP with GluA2-
R845A, an AMPAR mutant resistant to AP2-dependent endocytosis, has previously reversed
deficient synaptic NMDAR responses and blocked LTD (Hsieh et al., 2006). Given that
subneurotoxic concentrations of Aβ can also reduce early-phase LTP (E-LTP) (Chen et al.,
2000, 2002), it is possible that AMPAR deficits precede NMDAR deficits in E-LTP.
However, other research has highlighted that NMDARs are impaired in both E- and L-LTP
(Fernández-Fernández et al., 2015). The signalling pathways by which AMPARs and CaN are
involved in AD remain hypothetical.
Protein Kinases A and C
AMPARs may contribute to AD-related neurotoxicity via various PKA- and PKC-mediated
signalling pathways. For example, it has been suggested that the binding of soluble Aβ to β2-
adrenoceptors of the prefrontal cortex could enhance the activation of PKA via Gs-protein
pathways, contribute to GluA1 phosphorylation at S845 and, rather paradoxically, enhance
AMPAR-mediated EPSCs (Wang et al., 2010). Additionally, the atypical PKC, protein kinase
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Mζ, has been reported to accumulate in NFTs of limbic or medial temporal lobe structures
and, specifically, in abnormal neurites expressing GluA1 and GluA2 (Crary et al., 2006).
However, a PKA- and PKC-mediated mechanism by which AMPARs contribute to AD-
related neurotoxicity and is potentially very important is calcium dysregulation.
Liu et al. (2010b) observed that Aβ treatment significantly decreased the levels of membrane
GluA2 (see fig. 4A) and also raised the free cytosolic Ca2+ concentration ([Ca2+]i) in cultured
hippocampal neurons compared to Aβ-untreated neurons from P0 C57 mice. 5 μΜ nifedipine,
tetrodotoxin (TTX) and SKF 96365, which act to depress [Ca2+]i, increased the levels of
membrane GluA2, regardless of the presence of Aβ (see fig. 4B, 4C), whereas 1 μM Bay K
8644, an activator of L-type voltage-gated Ca2+ channels, significantly increased cytosolic
Ca2+ and decreased membrane GluA2 in both Aβ-treated and –untreated neurons. Aβ and Bay
K 8644 increased S880-GluA2 phosphorylation, whereas nifedipine, TTX and SKF 96365
decreased it (see fig. 4D). Furthermore, the PKC inhibitor, bisindolylmeimide I, prevented the
Aβ- and Bay K 8644-induced increase in S880-GluA2 phosphorylation and decrease in
membrane GluA2 levels (see fig. 4E). The study of Liu et al. (2010b) could have been
strengthened by measurements of AMPAR-mediated and NMDAR-mediated EPSCs,
LTP/LTD detection, detection of apoptotic markers, or by a cell count of apoptotic neurons or
total hippocampal neurons in the culture. The researchers propose that soluble Aβ exerts its
neurotoxic effects in part by downregulating cell-surface GluA2 via PKC-mediated S880
phosphorylation of GluA2. Nonetheless, it is also possible that other cell-surface AMPAR
subtypes could have been phosphorylated and undergone fluctuations.
In another study, Whitcomb et al. (2015) injected Aβ oligomers intracellularly via passive
diffusion from patch clamping into cultured hippocampal neurons from 6-8-day-old Wistar
rats and a rapid rise in the amplitude of AMPAR-mediated EPSCs was observed. The same
observation was made when an NMDAR antagonist D-AP-5 was applied, and NMDAR-
mediated EPSCs were unaffected by Aβ oligomer infusion. This effect occurred even when
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AMPAR-mediated EPSC stimulation was ceased for 15 minutes, a finding that allowed
Whitcomb et al. (2015) to support that the effect of the Aβ oligomers is independent of the
need
Fig. 4 Aβ mediates synaptic GluA2 removal via PKC-dependent phosphorylation of S880. Described
according to information from Liu et al. (2010b). Cultures of hippocampal neurons from P0 C57 were used at 14
DIV. They were treated with 1 μM Aβ40 or Aβ42 for 24 hours and their protein lysates were probed for cell-
surface GluA2 and pS880-GluA2 with anti-GluA2 and anti-pS880 antibodies. [Ca2+]i was measured with 2 μM
fluorescent indicator Fluo-4/AM. A) The effect of Aβ42 and scrambled Aβ42 on membrane GluA2 and total
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GluA2 immunoreactivities (I. R.) was evaluated by Western Blotting and the use of anti-GluA2 antibody, 4 and
24 hours after Aβ treatment. B) Effect of 5 μM nifedipine, 1 μM Aβ40 and 1 μM Aβ42 on Ca2+ fluorescence
changes (ΔF/F0) over time (min). C) Effect of 5 μM nifedipine, 1 μM Aβ40 and 1 μM Aβ42 on membrane GluA2
and total GluA2 I. R. D) Effect of 5 μM nifedipine, 1 μM Aβ40 and 1 μM Aβ42 on pS880-GluA2 I. R. E) Effect of
10 nM GFX, a PKC inhibitor, 1 μM Aβ40 and 1 μΜ Aβ42 on pS880, membrane GluA2 and total GluA2 I. R. One-
way analysis of variance; Least Significant Difference (LSD) post hoc test; Mean ± SEM; n=6 independent
incubations; * p<0.05. Adapted from Liu et al., 2010b.
to stimulate the AMPAR-mediated EPSCs. The postsynaptic application of the Ca2+ chelator
BAPTA prevented the increase in the amplitude of AMPAR-mediated EPSCs, as did the
application of Rp-cAMPS and H89, which are both used as PKA inhibitors. The amplitude
increase in AMPAR-mediated EPSCs was not abolished by the PKC inhibitors Ro 32-0432
and PKC19-31. The CaMKII inhibitor KN-62 initially does not abolish the increase in
AMPAR-mediated EPSC amplitude in the short term, but appears to do so in the long term. In
the last parts of their experiment, Whitcomb et al. (2015) knocked down GluA1 or GluA2 by
biolistic shRNA transfection and also made use of a GluA1 S845-phosphomutant that can’t be
phosphorylated at S845. A rapid rise in AMPAR-mediated EPSC amplitude was observed
with GluA2 shRNA transfection, but not with the GluA1 S845-phosphomutant. Application
of IEM 1460, the CP-AMPAR inhibitor, diminished the Aβ-induced increase in the amplitude
of AMPAR-mediated EPSCs and the use of biotinylation assays revealed that surface GluA1
levels increased under the effect of Aβ, whereas the levels of surface GluA2/3 remained
unaltered. Whitcomb et al. (2015) concluded that intracellular Aβ oligomers drive the
insertion of CP-AMPARs via PKA-dependent mechanisms.
A consensus regarding how membrane AMPAR subtypes fluctuate under the effect of Aβ or
pathological tau has yet to be reached. This is evident if both the results of Liu et al. (2010b)
and Whitcomb et al. (2015) are taken into consideration, as one may argue that GluA2 surface
expression decreases under the effect of Aβ, whereas the other will support the opposite. It
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may be that fluctuations in the levels of AMPARs are region-specific as well as stage specific,
as membrane preparations from the neocortex of dementia patients have revealed that GluA1
and GluA2/3 levels increase in mild cognitive impairment, decrease in early-stage AD and
increase again in severe-stage AD (Revett et al., 2013). From the combined results of Liu et
al. (2010b) and Whitcomb et al. (2015), it could also be speculated that a reduction of
membrane GluA2 levels, along with an increase in membrane CP-AMPARs, would favour
Ca2+ influx into neurons, which could potentially be associated with LTD and reduced
AMPAR-mediated EPSCs. However, in the case of PKA, enhancement, and not suppression,
of AMPAR-mediated EPSCs was observed. Given that both excitotoxicity and excess
intracellular calcium have been implicated in AD (Nguyen et al., 2015), the possibility that
AMPARs are partly involved in AD-related excitotoxicity should not be excluded. Indeed,
GluA2 levels in the entorhinal and hippocampus of AD patients have been found to decrease
prior to NFT formation (Weiss, 2011). In addition, decreased ADAR2 activity and decreased
RNA editing of GluA2 has also been acknowledged in the hippocampus of AD patients
(Gaisler-Salomon et al., 2014), an effect which should be associated with increased Ca2+
permeability through GluA2 receptors. Furthermore, it has been suggested that Aβ activates
NMDARs, with the resulting Ca2+ influx causing CaN activation, which in turn may
dephosphorylate and activate the striatal-enriched tyrosine phosphatase (STEP61) (Revett et
al., 2013). STEP61 may then increase the endocytosis of both NMDARs and AMPARs (Revett
et al., 2013), which comes to show that NMDAR and, potentially, CP-AMPAR activation,
may favour iGluR endocytosis via activation of CaN and STEP61. The enhanced AMPAR-
mediated EPSCs and Ca2+ dysregulation observed in the studies of this section may be able to
trigger additional signalling mechanisms that would then act to induce synaptic AMPAR
deficits.
Mitochondrial dysfunction and caspases
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Aβ-induced activation of glutamate receptors has been linked to mitochondrial dysfunction
(Alberdi et al., 2010). Alberdi et al. (2010) made use of hippocampal and entorhinal cortical
neurons from E18 Sprague-Dawley rat embryos at 8-10 DIV. Whole-cell recordings were
performed at a holding potential of -70 mV. They first confirmed that 1 μM Aβ can activate
NMDARs and AMPARs, as they observed inward currents in most of the neurons they used.
In the case of NMDARs, this effect was not abolished by 1 μM TTX or calcium-free
extracellular bath solution. They used 2 μΜ fura-2 AM to image cytosolic Ca2+ and they
observed that 5 μM Aβ caused a sustained [Ca2+]i increase through activation of NMDARs
and AMPARs. The increase in [Ca2+]i was nearly blocked by iGluR antagonists and EGTA,
but not by voltage-gated Ca2+ channel inhibitors, e.g. nifedipine and verapamil. [Ca2+]i was
also quantified with 5 μM fluo-4-AM in hippocampal-entorhinal cortical slices. 20 μM
oligomeric Aβ increased [Ca2+]i in organotypic slices, an effect that was blocked by
simultaneous inhibition of AMPARs and NMDARs. Reactive oxygen species were assayed
with 30 μM CM-H2DCFDA, mitochondrial Ca2+ uptake was assayed with 2 μM rhod2-AM,
cell death was quantified by uptake of propidium iodide (PI) in organotypic slices and the
release of lactate dehydrogenase in neuronal cultures, and the mitochondrial membrane
potential was assayed with 3 μM JC-1. Alberdi et al. (2010) concluded that NMDAR and
AMPAR can induce mitochondrial Ca2+ overload, oxidative stress and mitochondrial
membrane depolarisation, which can be prevented by caspase inhibitors, e.g. ZVAD-F and
Ac-DEVD-F, nuclear enzyme poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor DPQ, and
iGluR antagonists.
From the study of Alberdi et al. (2010), it can be assumed that Ca2+ dysregulation can be
caused by Aβ-induced NMDAR and AMPAR activation or overexcitation, which is then
followed by mitochondondrial dysfunction and subsequent activation of caspases and
apoptosis. This can, in turn, be followed by AMPAR signalling deficits (Chan et al., 1999;
Glazner et al., 2000; Li et al., 2010). Li et al. (2010) induced LTD by LFS (1 Hz for 900 s) in
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CA1 synapses of rat (3-4-week-old) hippocampal slices, which was not induced when the
slices were incubated with inhibitors of caspases-3 and -9, e.g. 2 μM DEVD-FMK, 2 μM
LEHD-FMK, when caspase-3 was knocked-out or when anti-apoptotic proteins, e.g. BcI-xL
and XIAP, were overexpressed via biolistic plasmid transfection. They also observed that
treating hippocampal neurons (18 DIV) with 50 μM NMDA resulted in the internalisation of
endogenous AMPARs, an effect that was abolished by DEVD-FMK, LEHD-FMK, BcI-xL
and XIAP overexpression, and caspase-3 KO. Immunoblotting and biotin-DEVD staining was
used to detect caspase-3 activity, which increased with NMDA treatment prolonged exposure
to staurosporine, an inducer of apoptosis. This increase in caspase-3 activity was blocked by
NMDAR antagonists, e.g. APV, by EGTA and by inhibitors of intracellular calcium release,
e.g. 2-APB and dantrolene. PI staining showed that treatment with 50 μM NMDA did not
induce apoptosis. These neurotoxic effects were linked to mitochondrial dysfunction, as the
levels of cytosolic cytochrome c, the proapoptotic molecule released from mitochondria,
increased with 50 μM NMDA treatment.
For their study, Chan et al. (1999) used human brain tissue from AD patients and normal
control patients, as well as embryonic rat hippocampal cell cultures (7-9 DIV). The extent of
apoptosis was determined with the fluorescent DNA-binding dye Hoechst 33342 based on
DNA morphology. Caspase activity was estimated with an assay that measures the activity of
interleukin-1β converting enzyme (ICE) and the release of 7-amino-4-methylcoumarin from
Ac-YVAD-AMC. Western Blotting and immunohistochemistry was used to probe for GluA1,
GluA2/3, GluA4, GluN1, GluN2A, ICE and activated caspase-3. Chan et al. (1999) found that
caspase activity was greater in hippocampal tissue of AD patients than that of control patients
(see fig. 5A, 5B) and Western Blot analysis showed that the amounts of GluA1, GluA2/3 and
GluA4 were significantly decreased in AD hippocampi, whereas the levels of GluN1 and
GluN2A were not significantly affected (see fig. 5C). They also demonstrated that exposure
of the rat hippocampal neurons to micromolar concentrations of Aβ25-35 led to the apoptosis of
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the cultured neurons. Treatment with Aβ25-35 or staurosporine resulted in the degradation of
GluA1, GluA2/3 and GluA4, whereas co-treatment with zVAD-FMK prevented the
degradation of the AMPAR subtypes. Aβ25-35 and staurosporine had no effect on the levels of
GluN1 and GluN2A.
Fig. 5 Caspase activation and AMPAR degradation in the brains of AD patients. Described according to
information from Chan et al. (1999). A. Hippocampal tissues from control patients (upper panel) and AD
patients (lower panel) were stained with an antibody against activated caspase-3. Greatly increased
immunoreactivity was noted in the brains of AD patients. B. Quantification of caspase activation via
measurement of ICE immunoreactivity (absorbance at 380-460 nm) over time (hours) in the hippocampi of AD
and control patients in the presence or absence of an ICE inhibitor. Mean ± SEM; P<0.05 by paired t test. C.
Western Blot analysis (50 μg protein/lane) of AMPAR and NMDAR subtypes from the hippocampi of AD and
control patients. GluA1, GluA2/3 and GluA4 levels were reduced in the brains of AD patients compared to those
of controlled patients, whereas GluN1 and GluN2A levels were not altered. Adapted from Chan et al., 1999.
Similar to the study of Chan et al. (1999), Glazner et al. (2000) observed that AMPARs are
degraded by caspases whereas NMDARs are spared, when trophic support is withdrawn from
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cultured rat hippocampal slices. Interestingly enough, Glazner et al. (2000) also observed
increased colocalisation of caspase-3 immunoreactivity and PSD-95 immunoreactivity in
dendrites and cell bodies. It has previously been supported that the Aβ-induced activation of
NMDARs can lead to the loss of the postsynaptic density protein PSD-95 in a manner
dependent on caspases-3 and -8 (Liu et al., 2010a). AD transgenic mice or oligomeric Aβ-
treated neurons display reduced levels of PSD-95, along with dendritic spine loss and surface
AMPAR removal, and overexpression of α1-takusan, a PSD-95-binding protein, can protect
from such effects (Tu et al., 2014). Therefore, it is also probable that AD-related AMPAR
dysfunction is mediated by glutamate-induced excitotoxicity via the caspase-dependent loss
of PSD-95.
A link between Aβ-induced excitotoxicity, synaptic AMPAR deficits and overall synaptic
depression arises from these studies, as excessive activation of NMDARs and AMPARs leads
to Ca2+ dysregulation and mitochondrial dysfunction, followed by caspase activation and
either endocytosis or cleavage of AMPARs. AMPAR subtypes present caspase cleavage sites
with at least one site being specific for caspase-3 (Chan et al., 1999), and thus caspase-driven
degradation of AMPARs in AD is possible. Although in the aforementioned studies it is
argued that AMPARs are exclusively depleted as a result of caspase activation, this has also
been noted to occur with NMDARs in AD transgenic mice (Calon et al., 2005). Given that
PSD-95 also binds GluN2 subtypes (Sheng and Sala, 2001), it is possible that surface
NMDARs are also decreased as a result of caspase-mediated PSD-95 loss. However, it was
shown recently that inhibition of Ca2+ channels with 2-APB in hippocampal slices and
cultured hippocampal neurons from APPswe/PS1ΔΕ9 C57BL/6 mice, i.e. mice that bear both
mutant APP and mutant PS1, reversed oligomeric Aβ-induced LTP deficits, blocked BAX
and caspase-3 hyperactivation, and also prevented the Aβ-induced reduction in levels of
membrane GluA1 and pS831-GluA1 (Hu et al., 2015). On the other hand, oligomeric Aβ, 2-
APB or the combination of both had no effect on the levels of surface GluN2A and GluN2B
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(Hu et al., 2015). Therefore, it is widely implied that AMPARs may be playing a more
prominent role than NMDARs via this particular neurotoxic mechanism.
APOE ε4, TARPs and phospholipid pathways
As was previously mentioned, decreased GluA2 RNA editing has been noted in the brains of
AD patients (Gaisler-Salomon et al., 2014). This finding was more profound in the
hippocampus of AD patients that carry the ε4 variant of the APOE gene (APOE ε4), and
previous research has shown that mouse cells expressing human APOE ε4 exhibit abnormal
membrane AMPAR expression (Gaisler-Salomon et al., 2014). Given that APOE ε4 is a major
genetic risk factor for AD, it is possible that unedited GluA2 could function as an AD
biomarker. However, AMPARs may be playing a greater role than that of inefficient RNA
editing when it comes to APOE ε4, which has also been correlated with increased levels of
plasma low density lipoproteins (LDL) (Hauser et al., 2011). The interaction of lipids with
TARPs acts to inhibit the binding of TARPs to PSD-95 and the subsequent synaptic
localisation of AMPARs (Opazo et al., 2010; Sumioka et al., 2010), and it is possible that
TARP-mediated trafficking of AMPARs is impaired in AD patients with APOE ε4 alleles and
abnormal plasma lipid profiles (Chang et al., 2012). APOE ε4 has also been implicated in the
sequestration of both AMPARs and NMDARs in intracellular compartments (Chen et al.,
2010). Additionally, low-density receptor-related protein 1 (LRP1) modulates synaptic
transmission and plasticity by forming complexes with NMDARs and GluA1 (Gan et al.,
2014). APOE ε4 is the only APOE isoform that has not been correlated with reduced surface
removal of LRP1 (Ramanathan et al., 2015) and it has also been suggested that its neurotoxic
effects, e.g. Aβ accumulation, are mediated via LRP1 (Gilat-Frenkel et al., 2014). It may be
that the interaction between LRP1 and GluA1 is impaired in the presence of Aβ (Gan et al.,
2014). Other mechanisms revolve around synaptojanin 1 (synj1) and phospholipase A2. For
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example, the phosphoinositol (4,5)-bisphosphate (PIP2) phosphatase, synj1, regulates clathrin-
mediated AMPAR internalisation, and reduced expression of synj1 may counteract Aβ1-42-
induced AMPAR internalisation (Di Paolo and Kim, 2011). Synj1 reduction has already been
shown to increase Aβ clearance and attenuate cognitive decline in AD transgenic mice (Zhu et
al., 2013). Finally, Aβ-induced activation of group IV phospholipase A2 and excess
production of arachidonic acid has been correlated with increased surface levels of AMPARs
and AMPAR-mediated excitotoxicity (Di Paolo and Kim, 2011). All the mechanisms
analysed in this section can be seen in figure 6.
Fig. 6 Proposed pathways for dysregulation of AMPAR function by Aβ, APOE ε4, lipids and phospholipid
metabolites. 1. Low editing of GluA2 that favours calcium dysregulation and excitotoxicity. 2. APOE ε4
promotes hyperlipidaemia. Plasma lipids inhibit PSD-95-TARP interaction and AMPAR clustering. 3. iGluRs
localise at the synapse with LRP1. 4. iGluR sequestration in intracellular compartments. 5. Increased synj1
activity that drives clathrin-mediated AMPAR endocytosis. 6. Excess group IV phospholipase A2 activity and
arachidonic acid promote AMPAR insertion and excitotoxicity. Created by Louloudis, 2016 (unpublished).
Discussion
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The roles of AMPAR dysfunction in AD remain elusive, as there is no easy conclusion that
can be drawn from the research reviewed in this dissertation. AMPAR currents were either
diminished in AD with NMDAR currents being unaffected (Gu et al., 2009) or vice versa
(Chen et al., 2002). Additionally, the levels of GluA2 in the synaptic membrane were either
reduced (Liu et al., 2010b) or left unaffected (Gu et al., 2009). Despite such controversial
observations, some of the key findings were that decreased phosphorylation of S831-GluA1
by CaMKII (Gu et al., 2009), increased phosphorylation of S880-GluA2 by PKC (Liu et al.,
2010b) and increased dephosphorylation of S845-GluA1 by CaN (Miller et al., 2014) are
ways by which the molecular pathology of AD can result in internalisation of synaptic
AMPARs. At the same time, increased phosphorylation of S845-GluA1 drives the insertion of
CP-AMPARs at the synaptic membrane (Whitcomb et al., 2015), which can then result in
Ca2+ influx in conjunction with NMDAR activation (Alberdi et al., 2010). This can then lead
to caspase activation and loss of synaptic AMPARs (Chan et al., 1999). Furthermore, the
major genetic risk factor for AD, APOE ε4, has been correlated with increased levels of
unedited GluA2 that render AMPARs permeable to Ca2+ (Gaisler-Salomon et al., 2014).
Finally, lipid metabolic pathways may contribute to AD pathology by disrupting AMPAR
trafficking (Chang et al., 2012).
There are various factors that could have influenced the outcome of the experimental studies
mentioned in this dissertation. For example, different Aβ conformations could have
contributed to AMPAR dysfunction and AD neurotoxicity via different ways (Deshpande et
al., 2006). A solution that contains Aβ monomers only may have no effect on GluA1 of
dendritic spines of hippocampal neurons (Rui et al., 2010). However, Aβ42 monomers, but not
Aβ40, reportedly reduce the amplitude of AMPAR-mediated EPSCs (Parameshwaran et al.,
2007). This may be due to Aβ42 monomers exhibiting rapid oligomerisation compared to other
Aβ monomeric species (Bitan et al., 2002). A more reliable approach would be to use
solutions that contain all possible conformations and sizes of Aβ, e.g. monomers, oligomers
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and fibrils. The mechanisms of AMPAR dysfunction in AD and the alterations in AMPAR
levels that result from them could be region- and stage-specific (Revett et al., 2013). AMPAR
dysfunction in AD may spread throughout the brain in a Braak stage-like manner, which is
summarised by Carter et al. (2004). Specifically, there are certain areas, e.g. CA2, CA3,
dentate gyrus, that are resistant to AMPAR dysfunction. In the vulnerable areas, e.g.
subiculum, GluA2 and GluA2/3 decrease in Braak stages I-II and then even more in stages
III-IV, whereas in the final two Braak stages, GluA2 and GluA2/3 levels are similar to those
of the first two stages. GluA1 levels remain unchanged throughout all stages. In light of this
theory, the validity of the studies by Gu et al. (2009) and Whitcomb et al. (2015), which
report that GluA1 levels are altered in AD with GluA2/3 left unchanged, may be questioned.
It also raises the possibility that Ca2+ dysregulation in AD is downstream, and not upstream,
of AMPAR downscaling, as GluA2 reduction favours Ca2+ influx and increased [Ca2+]i
(Carter et al., 2004). Therefore, there are various factors that ought to be considered when
studying the potential roles of AMPAR dysfunction in AD, e.g. Aβ solution, anatomical
region and AD stage.
In the present dissertation, AMPAR dysfunction seems to be occurring as a result of the
dysfunction of many other components in the synapse, e.g. CaMKII, CaN, and, in the study of
Alberdi et al. (2010), NMDARs were more strongly activated by Aβ than AMPARs. Thus,
AMPAR dysfunction may not necessarily be a causative event of AD, but a biomarker.
AMPAR potentiators have been tested in clinical trials against cognitive decline in AD. No
beneficial effect has been observed by the use of LY451395 (Chappell et al., 2007), as well as
that of oxiracetam, the structural analogue of piracetam (Gouliaev and Senning, 1994). The
cognitive enhancer piracetam has yielded a positive effect in AD patients (Gouliaev and
Senning, 1994). However, a recent clinical study demonstrates that piracetam has no
significant effect on its own against AD, but it is effective when it is combined with the
cholinesterase inhibitor, rivastigmine (Iranmanesh et al., 2013). Therefore, the role of
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AMPAR positive modulators in the treatment of AD is also poorly understood. As
memantine, an NMDAR antagonist, is already in use against AD, its efficacy could be studied
when it is administered along with a CP-AMPAR inhibitor, e.g. IEM1064. Finally, further
research is needed to better understand the mechanisms of AMPAR plasticity. According to
Chater and Goda (2014), efforts could be directed towards understanding how extrasynaptic
AMPARs are transferred to synaptic sites for LTP induction, how TARP function is
influenced by AMPAR subtype composition, where exactly AMPAR exo-endocytic recycling
takes place, and how this recycling process is influenced by presynaptic release mechanisms
of glutamate or AMPAR subtype composition. Understanding these aspects of AMPAR
plasticity would allow for speculation and study of additional roles of AMPAR dysfunction in
AD, and the development of potential treatments.
Acknowledgements
Special thanks go to Dr Ian Coombs for his comments and feedback on this manuscript.
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