neur3904 the potential roles of ampa receptor dysfunction in alzheimer's disease

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

G. Louloudis / NPP (2016) 1-34 NEUR3904

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

1


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.

2


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.

7


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


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.

10


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


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,

13


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

14


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

15


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

16


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

17


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

18


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

19


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

20


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

21


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

22


(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

23


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

24


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

25


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

26


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.

Bibliography

Alberdi E, Sánchez-Gómez MV, Cavaliere F, Pérez-Samartín A, Zugaza JL, Trullas R,

Domercq M, Matute C (2010) Amyloid beta oligomers induce Ca2+ dysregulation and

neuronal death through activation of ionotropic glutamate receptors. Cell Calcium

47:264–272.

Banke TG, Bowie D, Lee H-K, Huganir RL, Schousboe A, Traynelis SF (2000) Control of

GluR1 AMPA Receptor Function by cAMP-Dependent Protein Kinase. J Neurosci

20:89–102.

Barria A, Derkach V, Soderling T (1997) Identification of the Ca2+/calmodulin-dependent

protein kinase II regulatory phosphorylation site in the alpha-amino-3-hydroxyl-5-

methyl-4-isoxazole-propionate-type glutamate receptor. J Biol Chem 272:32727–32730.

27


Bats C, Groc L, Choquet D (2007) The interaction between Stargazin and PSD-95 regulates

AMPA receptor surface trafficking. Neuron 53:719–734.

Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, Teplow DB (2002) Amyloid

-protein (A ) assembly: A 40 and A 42 oligomerize through distinct pathways. Proc Natl

Acad Sci 100:330–335.

Bliss TVP, Cooke SF (2011) Long-term potentiation and long-term depression: a clinical

perspective. Clin (Sao Paulo, Brazil) 66 Suppl 1:3–17.

Calon F, Lim GP, Morihara T, Yang F, Ubeda O, Salem N, Frautschy SA, Cole GM (2005)

Dietary n-3 polyunsaturated fatty acid depletion activates caspases and decreases NMDA

receptors in the brain of a transgenic mouse model of Alzheimer’s disease. Eur J

Neurosci 22:617–626.

Carter TL, Rissman RA, Mishizen-Eberz AJ, Wolfe BB, Hamilton RL, Gandy S, Armstrong

DM (2004) Differential preservation of AMPA receptor subunits in the hippocampi of

Alzheimer’s disease patients according to Braak stage. Exp Neurol 187:299–309.

Chan SL, Griffin WS, Mattson MP (1999) Evidence for caspase-mediated cleavage of AMPA

receptor subunits in neuronal apoptosis and Alzheimer’s disease. J Neurosci Res 57:315–

323.

Chang PK-Y, Verbich D, McKinney RA (2012) AMPA receptors as drug targets in

neurological disease--advantages, caveats, and future outlook. Eur J Neurosci 35:1908–

1916.

Chappell AS, Gonzales C, Williams J, Witte MM, Mohs RC, Sperling R (2007) AMPA

potentiator treatment of cognitive deficits in Alzheimer disease. Neurology 68:1008–

1012.

Chater TE, Goda Y (2014) The role of AMPA receptors in postsynaptic mechanisms of

synaptic plasticity. Front Cell Neurosci 8:401.

Chen QS, Kagan BL, Hirakura Y, Xie CW (2000) Impairment of hippocampal long-term

28


potentiation by Alzheimer amyloid beta-peptides. J Neurosci Res 60:65–72.

Chen Q-S, Wei W-Z, Shimahara T, Xie C-W (2002) Alzheimer amyloid beta-peptide inhibits

the late phase of long-term potentiation through calcineurin-dependent mechanisms in

the hippocampal dentate gyrus. Neurobiol Learn Mem 77:354–371.

Chen Y, Durakoglugil MS, Xian X, Herz J (2010) ApoE4 reduces glutamate receptor function

and synaptic plasticity by selectively impairing ApoE receptor recycling. Proc Natl Acad

Sci U S A 107:12011–12016.

Crary JF, Shao CY, Mirra SS, Hernandez AI, Sacktor TC (2006) Atypical protein kinase C in

neurodegenerative disease I: PKMzeta aggregates with limbic neurofibrillary tangles and

AMPA receptors in Alzheimer disease. J Neuropathol Exp Neurol 65:319–326.

Deshpande A, Mina E, Glabe C, Busciglio J (2006) Different conformations of amyloid beta

induce neurotoxicity by distinct mechanisms in human cortical neurons. J Neurosci

26:6011–6018.

Di Paolo G, Kim T-W (2011) Linking lipids to Alzheimer’s disease: cholesterol and beyond.

Nat Rev Neurosci 12:284–296.

Dubois B et al. (2010) Revising the definition of Alzheimer’s disease: a new lexicon. Lancet

Neurol 9:1118–1127.

Ehrlich I, Malinow R (2004) Postsynaptic density 95 controls AMPA receptor incorporation

during long-term potentiation and experience-driven synaptic plasticity. J Neurosci

24:916–927.

Fernández-Fernández D, Dorner-Ciossek C, Kroker KS, Rosenbrock H (2015) Age-related

synaptic dysfunction in Tg2576 mice starts as a failure in early long-term potentiation

which develops into a full abolishment of late long-term potentiation. J Neurosci Res

94:266–281.

Fleming JJ, England PM (2010) AMPA receptors and synaptic plasticity: a chemist’s

perspective. Nat Chem Biol 6:89–97.

29


Gaisler-Salomon I, Kravitz E, Feiler Y, Safran M, Biegon A, Amariglio N, Rechavi G (2014)

Hippocampus-specific deficiency in RNA editing of GluA2 in Alzheimer’s disease.

Neurobiol Aging 35:1785–1791.

Gan M, Jiang P, McLean P, Kanekiyo T, Bu G (2014) Low-density lipoprotein receptor-

related protein 1 (LRP1) regulates the stability and function of GluA1 α-amino-3-

hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor in neurons. PLoS One

9:e113237.

Gilat-Frenkel M, Boehm-Cagan A, Liraz O, Xian X, Herz J, Michaelson DM (2014)

Involvement of the Apoer2 and Lrp1 receptors in mediating the pathological effects of

ApoE4 in vivo. Curr Alzheimer Res 11:549–557.

Glazner GW, Chan SL, Lu C, Mattson MP (2000) Caspase-Mediated Degradation of AMPA

Receptor Subunits: A Mechanism for Preventing Excitotoxic Necrosis and Ensuring

Apoptosis. J Neurosci 20:3641–3649.

Gouliaev AH, Senning A (1994) Piracetam and other structurally related nootropics. Brain

Res Rev 19:180–222.

Gu Z, Liu W, Yan Z (2009) {beta}-Amyloid impairs AMPA receptor trafficking and function

by reducing Ca2+/calmodulin-dependent protein kinase II synaptic distribution. J Biol

Chem 284:10639–10649.

Guo Q, Li H, Cole AL, Hur J-Y, Li Y, Zheng H (2013) Modeling Alzheimer’s disease in

mouse without mutant protein overexpression: cooperative and independent effects of

Aβ and tau. PLoS One 8:e80706.

Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer’s disease: progress and

problems on the road to therapeutics. Science 297:353–356.

Hauser PS, Narayanaswami V, Ryan RO (2011) Apolipoprotein E: from lipid transport to

neurobiology. Prog Lipid Res 50:62–74.

Hayashi Y (2000) Driving AMPA Receptors into Synapses by LTP and CaMKII:

30


Requirement for GluR1 and PDZ Domain Interaction. Science (80- ) 287:2262–2267.

He K, Lee A, Song L, Kanold PO, Lee H-K (2011) AMPA receptor subunit GluR1 (GluA1)

serine-845 site is involved in synaptic depression but not in spine shrinkage associated

with chemical long-term depression. J Neurophysiol 105:1897–1907.

Hsieh H, Boehm J, Sato C, Iwatsubo T, Tomita T, Sisodia S, Malinow R (2006) AMPAR

removal underlies Abeta-induced synaptic depression and dendritic spine loss. Neuron

52:831–843.

Hu W-Y, He Z-Y, Yang L-J, Zhang M, Xing D, Xiao Z-C (2015) The Ca(2+) channel

inhibitor 2-APB reverses β-amyloid-induced LTP deficit in hippocampus by blocking

BAX and caspase-3 hyperactivation. Br J Pharmacol 172:2273–2285.

Ikonomovic MD, Mizukami K, Davies P, Hamilton R, Sheffield R, Armstrong DM (1997)

The loss of GluR2(3) immunoreactivity precedes neurofibrillary tangle formation in the

entorhinal cortex and hippocampus of Alzheimer brains. J Neuropathol Exp Neurol

56:1018–1027.

Iranmanesh F, Vakilian A, Gadari F, Sayyadi A, Mehrabian M, Moradi M, Raesy E (2013)

Piracetam, Rivastigmine and Their Joint Consumption Effects on MMSE Score Status in

Patients with Alzheimer’s Disease. Iran J Pharmacol Ther 11:60–63.

Kim CH, Chung HJ, Lee HK, Huganir RL (2001) Interaction of the AMPA receptor subunit

GluR2/3 with PDZ domains regulates hippocampal long-term depression. Proc Natl

Acad Sci U S A 98:11725–11730.

Kim S, Violette CJ, Ziff EB (2015) Reduction of increased calcineurin activity rescues

impaired homeostatic synaptic plasticity in presenilin 1 M146V mutant. Neurobiol

Aging 36:3239–3246.

Kitazawa M, Medeiros R, Laferla FM (2012) Transgenic mouse models of Alzheimer disease:

developing a better model as a tool for therapeutic interventions. Curr Pharm Des

18:1131–1147.

31


Lacor PN, Buniel MC, Chang L, Fernandez SJ, Gong Y, Viola KL, Lambert MP, Velasco PT,

Bigio EH, Finch CE, Krafft GA, Klein WL (2004) Synaptic targeting by Alzheimer’s-

related amyloid beta oligomers. J Neurosci 24:10191–10200.

Lansdall CJ (2014) An effective treatment for Alzheimer’s disease must consider both

amyloid and tau. Biosci Horizons 7:hzu002–hzu002.

Li L, Stefan MI, Le Novère N (2012) Calcium input frequency, duration and amplitude

differentially modulate the relative activation of calcineurin and CaMKII. PLoS One

7:e43810.

Li Z, Jo J, Jia J-M, Lo S-C, Whitcomb DJ, Jiao S, Cho K, Sheng M (2010) Caspase-3

activation via mitochondria is required for long-term depression and AMPA receptor

internalization. Cell 141:859–871.

Lin D-T, Makino Y, Sharma K, Hayashi T, Neve R, Takamiya K, Huganir RL (2009)

Regulation of AMPA receptor extrasynaptic insertion by 4.1N, phosphorylation and

palmitoylation. Nat Neurosci 12:879–887.

Lisman J, Yasuda R, Raghavachari S (2012) Mechanisms of CaMKII action in long-term

potentiation. Nat Rev Neurosci 13:169–182.

Liu J, Chang L, Roselli F, Almeida OFX, Gao X, Wang X, Yew DT, Wu Y (2010a) Amyloid-

β induces caspase-dependent loss of PSD-95 and synaptophysin through NMDA

receptors. J Alzheimers Dis 22:541–556.

Liu S-J, Gasperini R, Foa L, Small DH (2010b) Amyloid-beta decreases cell-surface AMPA

receptors by increasing intracellular calcium and phosphorylation of GluR2. J

Alzheimers Dis 21:655–666.

Miller EC, Teravskis PJ, Dummer BW, Zhao X, Huganir RL, Liao D (2014) Tau

phosphorylation and tau mislocalization mediate soluble Aβ oligomer-induced AMPA

glutamate receptor signaling deficits. Eur J Neurosci 39:1214–1224.

Mucke L, Selkoe DJ (2012) Neurotoxicity of amyloid β-protein: synaptic and network

32


dysfunction. Cold Spring Harb Perspect Med 2:a006338.

Nakagawa T (2010) The biochemistry, ultrastructure, and subunit assembly mechanism of

AMPA receptors. Mol Neurobiol 42:161–184.

Nguyen L, Lucke-Wold BP, Mookerjee SA, Cavendish JZ, Robson MJ, Scandinaro AL,

Matsumoto RR (2015) Role of sigma-1 receptors in neurodegenerative diseases. J

Pharmacol Sci 127:17–29.

Opazo P, Labrecque S, Tigaret CM, Frouin A, Wiseman PW, De Koninck P, Choquet D

(2010) CaMKII triggers the diffusional trapping of surface AMPARs through

phosphorylation of stargazin. Neuron 67:239–252.

Parameshwaran K, Sims C, Kanju P, Vaithianathan T, Shonesy BC, Dhanasekaran M, Bahr

BA, Suppiramaniam V (2007) Amyloid beta-peptide Abeta(1-42) but not Abeta(1-40)

attenuates synaptic AMPA receptor function. Synapse 61:367–374.

Ramanathan A, Nelson AR, Sagare AP, Zlokovic B V (2015) Impaired vascular-mediated

clearance of brain amyloid beta in Alzheimer’s disease: the role, regulation and

restoration of LRP1. Front Aging Neurosci 7:136.

Reese LC, Taglialatela G (2011) A role for calcineurin in Alzheimer’s disease. Curr

Neuropharmacol 9:685–692.

Regan P, Piers T, Yi J-H, Kim D-H, Huh S, Park SJ, Ryu JH, Whitcomb DJ, Cho K (2015)

Tau phosphorylation at serine 396 residue is required for hippocampal LTD. J Neurosci

35:4804–4812.

Revett TJ, Baker GB, Jhamandas J, Kar S (2013) Glutamate system, amyloid ß peptides and

tau protein: functional interrelationships and relevance to Alzheimer disease pathology. J

Psychiatry Neurosci 38:6–23.

Rui Y, Gu J, Yu K, Hartzell HC, Zheng JQ (2010) Inhibition of AMPA receptor trafficking at

hippocampal synapses by beta-amyloid oligomers: the mitochondrial contribution. Mol

Brain 3:10.

33


Shankar GM, Walsh DM (2009) Alzheimer’s disease: synaptic dysfunction and Abeta. Mol

Neurodegener 4:48.

Sheng M, Sala C (2001) PDZ domains and the organization of supramolecular complexes.

Annu Rev Neurosci 24:1–29.

Sumioka A, Yan D, Tomita S (2010) TARP phosphorylation regulates synaptic AMPA

receptors through lipid bilayers. Neuron 66:755–767.

Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R, Hansen LA, Katzman R

(1991) Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the

major correlate of cognitive impairment. Ann Neurol 30:572–580.

Tu S, Okamoto S, Lipton SA, Xu H (2014) Oligomeric Aβ-induced synaptic dysfunction in

Alzheimer’s disease. Mol Neurodegener 9:48.

Wang D, Govindaiah G, Liu R, De Arcangelis V, Cox CL, Xiang YK (2010) Binding of

amyloid beta peptide to beta2 adrenergic receptor induces PKA-dependent AMPA

receptor hyperactivity. FASEB J 24:3511–3521.

Weiss JH (2011) Ca permeable AMPA channels in diseases of the nervous system. Front Mol

Neurosci 4:42.

Whitcomb DJ, Hogg EL, Regan P, Piers T, Narayan P, Whitehead G, Winters BL, Kim D-H,

Kim E, St George-Hyslop P, Klenerman D, Collingridge GL, Jo J, Cho K (2015)

Intracellular oligomeric amyloid-beta rapidly regulates GluA1 subunit of AMPA

receptor in the hippocampus. Sci Rep 5:10934.

Wright A, Vissel B (2012) The essential role of AMPA receptor GluR2 subunit RNA editing

in the normal and diseased brain. Front Mol Neurosci 5:34.

Xue B, Edwards MC, Mao L-M, Guo M-L, Jin D-Z, Fibuch EE, Wang JQ (2014) Rapid and

sustained GluA1 S845 phosphorylation in synaptic and extrasynaptic locations in the rat

forebrain following amphetamine administration. Neurochem Int 64:48–54.

Zhao D, Watson JB, Xie C-W (2004) Amyloid beta prevents activation of

34


calcium/calmodulin-dependent protein kinase II and AMPA receptor phosphorylation

during hippocampal long-term potentiation. J Neurophysiol 92:2853–2858.

Zhao W-Q, Santini F, Breese R, Ross D, Zhang XD, Stone DJ, Ferrer M, Townsend M, Wolfe

AL, Seager MA, Kinney GG, Shughrue PJ, Ray WJ (2010) Inhibition of calcineurin-

mediated endocytosis and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

(AMPA) receptors prevents amyloid beta oligomer-induced synaptic disruption. J Biol

Chem 285:7619–7632.

Zhu L, Zhong M, Zhao J, Rhee H, Caesar I, Knight EM, Volpicelli-Daley L, Bustos V, Netzer

W, Liu L, Lucast L, Ehrlich ME, Robakis NK, Gandy SE, Cai D (2013) Reduction of

synaptojanin 1 accelerates Aβ clearance and attenuates cognitive deterioration in an

Alzheimer mouse model. J Biol Chem 288:32050–32063.

35

neur3904 the potential roles of ampa receptor dysfunction in alzheimer's disease

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