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Novel cyclic AMP signalling avenues in learning and memoryOstroveanu, A

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Chapter 7

General Discussion

Chapter 7

Content

1. The role of A-kinase anchoring protein 150 in learning and memory

2. PKA anchoring to AKAPs in learning and memory

3. Tools to investigate the function of AKAP(s) signalosome

4. mAKAP in the mouse brain

5. AKAPs as new therapeutic targets

6. The role of Epac in learning and memory

7. Overall conclusion and future perspectives

8. Reference

142

General discussion

1. The role of A-kinase anchoring protein 150 (AKAP150) in

learning and memory

Recently, A-kinase anchoring proteins (AKAP) emerged as prototypic coordinators of

cyclic adenosine monophosphate (cAMP) activated protein kinase (PKA) signaling

providing high specificity to PKA signaling. The most investigated member of this family

of proteins is AKAP79/150, a protein expressed in e.g. rodent (AKAP150) and human

(AKAP79) brain. We showed that AKAP150 is highly expressed in the mouse striatum,

cerebral cortex, and forebrain regions, while a relatively high expression was found in the

hippocampus and olfactory bulb of the mouse brain. Low/no expression was observed in

the cerebellum, hypothalamus, thalamus, and brain stem (Ostroveanu et al., 2007). When

the AKAP150 staining in the mouse brain is compared to the previously reported

AKAP150 immunoreactivity pattern in rats (Glantz et al., 1992, Lilly et al., 2005), it

becomes obvious that the AKAP150 distribution in rat brain is much more homogeneous

than in the mouse brain. For example, in mice the CA3 area of the hippocampus is very

densely stained compared to the remainder of the hippocampus (Ostroveanu et al., 2007),

while the rat hippocampus shows a homogenous staining. Overall, the expression patterns

of AKAP150 in the mouse and rat brain reveal an abundant presence in brain regions

involved in learning and memory such as the cortex and hippocampus. Although it is

difficult to speculate on the role of AKAP150 in the mouse brain on the basis of its

distribution, these data suggest that AKAP150 may well play a role in learning and

memory.

In a study focusing on the distribution of AKAP150 at rat CA1 pyramidal cell postsynaptic

densities (PSD), it was shown that AKAP150 interacts with components of the excitatory

PSD, whereas AKAP150 immunoreactivity (IR) was not associated with inhibitory

synapses (Lilly et al., 2005). In parallel to these findings, AKAP79/150 was identified as a

constituent of postsynaptic densities (PSD) of excitatory synapses in human cerebral cortex

(Carr et al., 1992). Moreover, AKAP79/150 is linked to the N- methyl D-aspartate receptor

(NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)

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Chapter 7

in the PSD through binding with postsynaptic density-95, discs large, zona occludens-1

(PDZ) domain, membrane-associated guanylate kinase (MAGUK), scaffold proteins

postsynaptic density-95 (PSD-95) and synapse-associated protein-97 (SAP97) (Colledge et

al., 2000). In addition to PKA, AKAP79/150 can also bind protein phosphatase 2B (PP2B)

and protein kinase C (PKC) (Carr et al., 1992b; Coghlan et al., 1995; Klauck et al., 1996;

Sik et al., 2000). It was found that this multi-enzyme signaling complex plays an important

role in coordinating changes in synaptic structure and receptor signaling functions

underlying synaptic plasticity (Dell'Acqua et al., 2006). For example, AKAP79/150 bound

PKA was found to be essential for the regulation of AMPA receptor surface expression and

synaptic plasticity (Rosenmund et al., 1994). Additional evidence that AKAP79/150 could

be implicated in synaptic transmission arises from the study of Genin and colleagues (2003)

who showed that AKAP150 mRNA is upregulated during the maintenance phase of long-

term potentiation (LTP), a mechanism of long-lasting enhancement in synaptic efficacy

(Genin et al., 2003). A very recent study by Lu et al. (2007) also suggested that AKAP150

can be involved in synaptic plasticity. In transgenic mice expressing a truncated form of

AKAP150 which cannot anchor PKA, hippocampal LTP was abolished in 7–12 but not 4-

week-old mice. From these data they concluded that PKA anchored to AKAP150 critically

contributes to LTP in the adult hippocampus (Lu et al, 2007). Changes in synaptic plasticity

are suggested to be the mechanism underlying learning and memory processes (Martin et

al., 2000; Micheau and Riedel 1999). The findings that AKAP79/150 plays an important

role in synaptic plasticity, thus, provide additional evidence for the hypothesis that

AKAP79/150 is important in learning and memory processes. However, knowledge on the

role of AKAP79/150 in learning and memory remained so far rather limited.

Initial evidence for a role of AKAPs and PKA anchoring in learning and memory came

from a study by Moita and colleagues. They blocked PKA anchoring in the lateral

amygdala of rats and subjected the animals to auditory fear conditioning (Moita et al.,

2002). Their data showed that inhibition of PKA binding to AKAPs in the rat lateral

amygdala impairs memory consolidation during auditory fear conditioning (Moita et al.,

2002). We provided the first direct evidence for a role of AKAP150 in learning and

memory processes by assessing the expression of AKAP150 in the mouse hippocampus

144

General discussion

after a single training session in a contextual fear conditioning paradigm. We could show

for the first time that AKAP150 is upregulated in the mouse hippocampus 6 hours after

training in this fear conditioning test (Nijholt et al., 2007). The time point of 6 h correspond

to the late phase of memory consolidation and is critically regulated by cAMP signaling

pathways in fear motivated associative learning. Moreover, mice exposed to a novel context

also showed an AKAP150 upregulation in the mouse hippocampus (Nijholt et al., 2007).

Although we can only speculate on the possible role of the increased expression of

AKAP150, it might very well be that elevated AKAP150 levels result in a more efficient

propagation of signals carried by locally generated cAMP (Colledge et al., 2000; Feliciello,

et al., 1997), which in turn may contribute to processing the exposure to a novel context and

the consolidation of associative memory. Since AKAP150 levels were only measured 6 h

after training we cannot exclude that AKAP150 may also be important during other stages

of the memory process. In general, the upregulation of AKAP150 may be the result of de

novo protein synthesis or decreased protein degradation.

Recently several studies provided data on possible mechanism of how AKAP150 could

modulate learning and memory processes. It was shown that constitutive loss of AKAP150

in mice modifies excitatory synaptic transmission (Tunquist et al., 2008). This is achieved

by a deranged localization of PKA in AKAP150 null hippocampal neurons which in turn

modulates the postsynaptic AMPA receptors. These AKAP150 KO mice also exhibit

deficits in motor coordination and show memory retrieval impairment in the Morris water

maze (Tunquist et al., 2008). Hall and colleagues (2007) showed that AKAP150 copurifies

with L-type Ca2+ channels Cav1.2 in the rat forebrain. Cav1.2 constitutes 80% of the L-type

channels in the brain (Hell et al, 1993, Moosmang et al., 2005) and is concentrated at

postsynaptic sites (Hell et al., 1996). It has been shown that hippocampal pyramidal

neurons express predominantly the Cav1.2 channel (Hell et al., 1993, Davare et al., 2001,

Sinnegger-Brauns et al., 2004). Interestingly, conditional inactivation of the Cav1.2 gene in

the mouse hippocampus and neocortex leads to a selective loss of protein synthesis-

dependent NMDAR-independent Schaffer collateral/CA1 late-phase LTP and these mice

show a severe impairment in a water-maze spatial-discrimination task (Moosmang et al,

2005). Although it was shown that Cav1.2 binds several AKAPs in the brain (MAP2B

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Chapter 7

(Davare et al., 1999, Hall et al., 2007), AKAP15 (Hall et al., 2007)), AKAP150 proved to

be critical for PKA-mediated regulation of this channel in the brain (Hall et al., 2007).

Overall, these data suggest that AKAP150 could be important in learning and memory via

the regulation of Ca2+ signaling through Cav1.2.

In addition, Chai et al. found evidence that AKAP150 co-immunoprecipitates with the acid-

sensing ion channels ASIC1a and ASIC2a (Chai et al., 2007). It has been shown that these

channels are critically regulated by AKAP150 and calcineurin/PP2B (Chai et al., 2007).

Interestingly, ASIC1a is abundantly expressed in the amygdala complex and other brain

regions known to be involved in fear memory, and has been implicated in LTP (Wemmie et

al., 2002, Chai et al., 2007). Moreover, ASIC1 null mice display deficits in cued and

contextual fear conditioning (Wemmie et al., 2003), whereas ASIC1a overexpression

enhances fear-related freezing behavior (Wemmie et al., 2004). Furthermore, inhibition of

PKA anchoring by Ht-31 induces a decrease in ASIC current amplitude in cultured mouse

cortical neurons and Chinese hamster ovary (CHO) cells (Chai et al., 2007). Altogether

these studies suggest that AKAP150 may be able to change memory performance via the

regulation of ASIC channels.

Recently, several other AKAPs were reported to be important contributors to learning and

memory processes. For example, it has been shown that AKAP Yu is involved in the

formation of long term memory in Pavlovian olfactory learning of Drosophila (Lu et al.,

2007) and WAVE-1 null-mice (the first AKAP null mouse model) are impaired in spatial

(Morris water maze) and non-spatial (novel object recognition) memory formation but not

in emotional learning and memory (passive avoidance) (Soderling et al., 2003).

In summary, these data provide evidence for an increasing number of AKAPs that are

involved in learning and memory in different species. Our data, together with future

findings will lead to the understanding of how AKAPs coordinate the action of signaling

networks in cognitive processes under physiological and pathological conditions.

Ultimately this knowledge on the coordination of signaling cascades may lead to the

development of novel, more fine-tuned, innovative therapeutic strategies to treat cognitive

dysfunction.

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General discussion

2. PKA anchoring to AKAPs in learning and memory

Besides the role of particular AKAPs in learning and memory, it is at least as important to

have a good understanding of the role of enzyme binding to these AKAPs. We addressed

the question whether anchored PKA is critically involved in the different stages

(acquisition, consolidation, retrieval, and extinction) of contextual fear memory in mice.

Accordingly, we first injected mice intracerebroventricularly with St-Ht31 (a membrane

permeable peptide that competes for PKA anchoring) at different time points during the

memory process. Injecting mice before training with St-Ht31 to affect the acquisition phase

resulted in impaired associative fear memory 24 h after the training. Similarly, injection of

St-Ht31 immediately after training (consolidation phase) significantly attenuated

conditioned fear. The learning deficit observed when St-Ht31 was injected immediately

after training was similar to the effect of St-Ht31 injected before training. To discriminate

between acquisition and consolidation, we also performed the retention test 1 h after the

training with mice that were injected before the training. Interestingly, the performance of

St-Ht31 injected animals did not differ from the control groups when the retention test was

performed 1 h after training. The finding that mice which received St-Ht31 before training,

showed unimpaired freezing 1 h after training but attenuated freezing 24 h after training,

suggests that PKA anchoring onto AKAPs plays a specific role in the consolidation of

contextual fear memories but not in acquisition (Nijholt et al., 2008). Moreover, disrupting

PKA anchoring before the retention test had no effect on memory performance in

contextual fear conditioning (Nijholt et al., 2008).

Next, we tested the sub-region specific contribution of PKA anchoring in the hippocampus

to the consolidation phase of fear memory. Therefore we injected immediately after training

St-Ht31 locally in the CA1 area of the mouse hippocampus. During the retention test mice

showed reduced freezing behavior when compared to vehicle or non-injected animals,

which indicates that disrupting PKA anchoring locally in the CA1 is instrumental in the

consolidation of fear memory. Our finding that PKA anchoring is important in learning is

supported by the findings of Nie and colleagues (Nie et al., 2007). Genetically modified

mice conditionally expressing Ht31 in the forebrain regions show impairments in the spatial

version of the Morris water maze, a hippocampus-dependent memory task (Nie et al, 2007).

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Although these data showed that PKA anchoring is crucial for memory consolidation, they

did not provide information on whether type I and/or type II PKA is involved. However,

most of the AKAPs in the brain tether PKA type II. To date, no RI AKAP was described in

the brain and only a few dual RI/RII AKAPs were characterized (Huang et al, 1997). Since

we cannot completely rule out a role for RI anchoring, we injected stearated Super-AKAP-

IS, in the mouse CA1 area of hippocampus before training mice in contextual fear

conditioning. Super-AKAP-IS is a PKA anchoring inhibitory peptide that selectively binds

RIIβ subunits of PKA. This selective anchoring inhibitory peptide also impaired the

freezing behavior when injected immediately after training. Thus, RIIβ anchoring to

AKAPs in the CA1 of the mouse hippocampus is crucial and sufficient for the

consolidation of contextual fear memory.

Recently Isiegas and colleagues showed that neuronal PKA facilitates the extinction of

contextual fear (Isiegas et al., 2006). This was found in transgenic mice expressing from

birth a dominant-negative form of PKA and in mice in which the same PKA dominant-

negative form was temporarily regulated in brain regions thought to be involved in

extinction (Isiegas et al., 2006). In our lab, we assessed for the first time the role of PKA

anchoring in fear extinction. Mice were injected in the CA1 area of the hippocampus with

stearated Super-AKAP-IS immediately after each extinction trial of contextual fear

memory. We observed that inhibiting PKA anchoring in the hippocampus promotes the

extinction of contextual fear conditioning (Nijholt et al., 2008). We thus were the first to

show that disrupting RIIβ anchoring only in the hippocampus is sufficient to facilitate fear

extinction. Our data are supported by the finding that temporally transgenic inhibition of

PKA activity also promotes extinction (Isiegas et al., 2006). More evidence for a role of

PKA anchoring in memory formation was provided by studies in Drosophila where AKAP-

bound PKA is required for aversive memory during appetitive memory, a Pavlovian

olfactory learning task in which an electric shock is used as an aversive unconditioned

stimulus (Schwaerzel et al., 2007). Unfortunately, St-Ht31 and Super-AKAP-IS do not

specifically block PKA anchoring to a particular AKAP, and therefore it cannot be

concluded which AKAP(s) was involved in the impaired consolidation of contextual fear

memory. We thus determined the amount of PKA anchored to AKAP150 in the dorsal

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General discussion

hippocampus after intrahippocampal injection of St-superAKAP-IS. St-superAKAP-IS was

shown to reduce the amount of PKA anchored to AKAP150 in the dorsal hippocampus

when compared to vehicle-injected mice.

It was reported that PKA bound to AKAP150 is crucial for the phosphorylation of GluR1

AMPA receptor subunit at Ser845 (Colledge et al., 2000, Tavalin et al, 2002). This

phosphorylation stimulates postsynaptic accumulation of GluR1-containing AMPARs

during LTP (Esteban et al, 2003; Oh et al, 2006). Previously, it has been shown that

treatment of hippocampal slices with membrane permeable Ht31 impairs PKA dependent

hippocampal late-phase LTP (L-LTP) (Huang et al., 2006). Recently, Nie and colleagues

showed in transgenic mice expressing Ht31 in the hippocampus that a disruption of PKA-

AKAP interactions does not alter PKA activity or basal synaptic transmission but that only

long lasting forms of hippocampal LTP are impaired (Nie et al, 2007). Interestingly,

disruption of PKA anchoring or inhibition of PKA activity in hippocampal neurons leads to

a PP2B dependent, long-term depression (LTD) -like downregulation of AMPAR currents

and loss of AMPAR surface expression that likely involves AKAP79/150 (Hoshi et al.,

2005; Rosenmund et al., 1994; Snyder et al., 2005; Tavalin et al., 2002). Overall, these data

suggest that AKAP79/150 regulates the balance between PKA and PP2B signaling to

control AMPAR phosphorylation underlying LTP and LTD in synaptic plasticity. However,

so far the dynamics of this process are poorly understood. NMDA treatments that induce

LTD at many synapses simultaneously (chem-LTD) in hippocampal neurons activate

PP2B/CaN signaling and disrupt the association of AKAP79/150 with PSD-95 and

cadherins. This leads to a loss of the AKAP from spines and coincides with the removal of

AMPA receptors from synapses (Gomez et al., 2002; Gorski et al., 2005). In hippocampal

slices, chem-LTD activation of PP2B/CaN is followed by a persistent redistribution of both

AKAP79/150 and PKA-RII from postsynaptic membranes to the cytoplasm, without

PP2B/CaN translocation (Smith et al., 2006). Furthermore, using fluorescence resonance

energy transfer microscopy in hippocampal neurons, Smith et al. showed that PKA

anchoring to AKAP79/150 is required for an NMDA receptor-dependent regulated

cytoplasmic translocation of PKA and AKAP79/150 (Smith et al., 2006).

In summary, PKA anchoring in the hippocampus is required for both long-lasting forms of

hippocampal synaptic plasticity, and learning and memory.

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3. Tools to investigate the function of AKAP(s) signalosome

Besides PKA, there are more AKAP binding partners that participate in learning and

memory processes. For example, protein phosphatases are already widely acknowledged as

key molecules in synaptic plasticity and learning and memory. However, the role of protein

phosphatase anchoring to AKAPs in learning and memory has not been investigated yet.

The most interesting way to explore the function of any AKAP signalosome is by

pharmacological studies and/or by genetic manipulation. Although the use of

pharmacological compounds that block PKA anchoring already yielded fundamental

insights into the role of AKAP signaling in various physiological systems, inhibitory

peptides for any other AKAP binding partners are still not available to date. This is most

likely due to the difficulty of designing such agents and of intracellular delivery, and to the

expected unspecific action of such peptides.

In future experiments the impact of the RI isoform-selective anchoring on learning and

memory processes could be assessed using the RI anchoring disruptor (RIAD) (Carlson et

al., 2006). To study in greater detail which specific AKAP is involved, it would be

necessary to develop inhibitors that disrupt the interaction of PKA with one particular

AKAP or to disrupt the interaction of PKA by introducing site-specific mutations in the

PKA binding domain of a specific AKAP. Recently, genetically modified mice were

generated in the AKAP field. It is not surprising that the first complete knockdown of an

AKAP was AKAP150 (Hall et al., 2007). In addition to the AKAP150 KO mouse, mice in

which the PKA binding site was removed from the AKAP150 gene were also generated

(Hall et al., 2007). However, these mice are still not perfect “tools” for investigating the

role of AKAP150 since the modification occurs also during development and compensatory

mechanisms cannot be overcome. To define a role for a specific AKAP in learning and

memory, precise control on the expression of the transgene in space and time is required.

Another advantage of this spatial inducible system, compared to constitutive deletion of a

gene or expression of a transgene, is a reduced chance of animal lethality. This is especially

relevant when the gene of interest plays a crucial role during development or encodes for a

toxic protein. Already, the tetracycline-dependent system is widely used as a tool for spatio-

temporal control of transgene expression. Using this system, Isiegas and coworkers

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General discussion

generated genetically modified mice that conditionally express Ht31 in forebrain regions.

Behavioral studies with these Ht31 transgenic mice revealed impairments in spatial learning

and memory in the Morris water maze as well impairments in long-lasting forms of

hippocampal LTP (Isiegas et al., 2008).

In our lab we aimed to generate mice that inducibly express AKAP150 or AKAP150

mutants only in forebrain regions. In order to explore both the in vitro and in vivo relevance

of AKAP150 binding to PKA and PP2B, particularly in learning and memory, a strategy

was developed involving removal of the PKA and PP2B binding site from AKAP150.

Initially, using a polymerase chain reaction (PCR) based technique we were able to clone

the mouse AKAP150 protein. Later on, by comparing the mouse AKAP150 sequence with

the human AKAP79 and rat AKAP150 homologues we could identify the PKA and PP2B

binding site on mouse AKAP150 and generate several deletion mutants: the PKA binding

site deletion mutant, the PP2B binding site deletion mutant, and the PKA+PP2B binding

site deletion mutant (Fig. 1).

Fig. 1. Left panel. Schematic

diagram of the AKAP150

molecule. The PKA and PP2B

binding site are located near the C-

terminal end of the protein. The N-

terminal end of the protein

contains a binding site for PKC

and the membrane-targeting

domain. Right panel. Several mutants were generated: upper - binding site-deleting mutant for PKA, middle -

binding site-deleting mutant for PP2B, lower - binding site-deleting mutant for both PKA and PP2B

To generate mice with an AKAP150 overexpression or an AKAP150 mutant

overexpression we opted for the use of lentiviral vectors to transduce embryonic stem cells.

These stem cells can then be injected into a mouse blastocyst to create a chimerical

transgenic animal. Later on, by cross breeding of chimeric transgenic animals, a pure

transgenic animal can be obtained. These transgenic animals can then be used for

functional testing to determine the consequences of their altered genetic material (Fig. 2).

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Moreover, by using the lentiviral expression system, various cell lines could be infected in

addition to test the viability and function of AKAP150 and its mutants in vitro. We

managed to successfully infect embryonic stem cells with AKAP150 or AKAP150 binding

site deletion mutants (unpublished data). These results will allow us to move forward in

creating a transgenic animal expressing inducible AKAP150 binding site deletion mutants

in the near future. Unquestionably, these transgenic animals can be used in future

experiments for a better understanding of the role of AKAP150 and anchoring of PKA and

PP2B to AKAP150 in learning and memory.

GENE OF INTEREST

Virus producer cell line

Producing

viral particle

Lentiviral vector

+

Gene of interest Overexpress gene of interest

by infecting the cell line of interest

In vitro experiments

+

Infecting embryonic stem cells

Select for cells

expressing

desired gene

Inject transformed ES

cells into blastocyst

Transgenic animal

Behavioral

testing

Electrophysiology

Fig. 2. Flowchart of the lentiviral expression system. The genes of interested were cloned in lentiviral expression

vectors. Viral particles containing the transgene can be use to infect various cell lines. This will allow for in vitro

experiment to test the function of the wildtype and mutants. Viral particles can also be used to infect embryonic

stem cells that can be used to generate transgenic animals. The physiological relevance of mutant AKAP150 in

transgenic animals can be assessed by behavioral testing, in vitro experiments, and electrophysiology.

4. mAKAP in the mouse brain

In the last few years, the number of AKAPs that were identified and characterized increased

considerably. One of these, mAKAP (m of muscular), was found to be predominantly

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General discussion

expressed in cardiac and skeletal muscle, but also detected to some extent in the brain

(Kapiloff et al., 1999). Two alternatively spliced variants of mAKAP were subsequently

characterized: mAKAPα (preferentially expressed in the brain) and mAKAPβ (abundant in

cardiac myocytes and skeletal muscle). In addition to their tissue-specific expression,

mAKAPα and mAKAPβ considerably differ in size: the longer form mAKAPα contains an

additional 244 amino acid residue N-terminal extension (Michel et al., 2005). Detailed

knowledge on the localization of mAKAP in the brain remained so far limited. We showed

that mAKAP is abundantly expressed throughout the entire mouse brain (Ostroveanu et al.,

2009 submitted). Both immunohistochemistry and Western blotting revealed a high

expression of mAKAP in the cortex, cerebellum, thalamus, hypothalamus, and

hippocampus, while the lowest mAKAP expression was detected in the brain stem.

Moreover, at subcellular levels, mAKAP was abundantly expressed both in perikarya (e.g:

pyramidal neurons, Purkinje neurons), dendrites (e.g.: apical and basal dendrites of

hippocampus pyramidal neurons) and to some extent in fibers (e.g: amygdala, cortex). Our

results are in good agreement with the reported mRNA expression of AKAP100/mAKAP

in several human brain regions (Kapiloff et. al., 1999; McCartney et al., 1995).

The function of mAKAP in the brain still remains rather unexplored. Conversely, in the

heart the function of mAKAP was extensively studied. In the heart, mAKAP is targeted to

the nuclear envelope through the binding of three spectrin repeat domains, where it forms a

large macromolecular signaling complex containing several signal transduction molecules,

including PKA (Kapiloff et al, 1999, Kapiloff et al, 2001), the phosphodiesterase PDE4D3

(Dodge et al., 2001), the protein phosphatases PP2A and calcineurin (Kapiloff et al, 2001,

Pare et al., 2005), ryanodine receptors (RyR2) (Kapiloff et al, 2001, Marx et al., 2000,

Ruehr et al., 2003), the small GTPase Rap1, the guanine exchange factor Epac1 (Dodge-

Kafka et al., 2005), and the mitogen-activated protein kinases (MAPK) MEK5 and ERK5

(Dodge-Kafka et al., 2005). Both in the brain and heart, mAKAP can also bind 3’-

phosphoinositide-dependent kinase 1 (PDK1) and p90 ribosomal S6 kinase (RSK3) (Michel

et al., 2005). Although in the heart 12 mAKAPβ signalosome components were reported,

there are probably many more mAKAPβ binding partners.

The mAKAPα signalosome in the brain has not been characterized yet. It can be expected

that mAKAPα and mAKAPβ form more or less the same signalosome. Preliminary data

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from our laboratory show that mAKAPα co-immunoprecipitates with PDE4D3 and the

exchange protein directly activated by cAMP 2 (Epac2) from hippocampal lysates

(unpublished data). Whether Epac1 is also included in the mAKAPα macromolecular

complex, still needs to be determined. In addition, preliminary colocalization studies in

primary cortical neurons confirmed that mAKAP co-localizes with Epac2 and PKA-RIIβ

(Fig. 3; unpublished data).

mAKAP PKA-RIIβ Merge

mAKAP Epac2 Merge

Fig. 3. mAKAP colocalizes with Epac2 and PKA-RIIβ in primary cortical neurons. Primary cortical neurons from

E14 were probed with antibodies against mAKAP, Epac2 and PKA-RIIβ.

Since at the nuclear membrane of cardiomyocytes the mAKAP complex coordinates three

different cAMP effectors (PKA, PDE4D3 and Epac1), and also calcium and MAPK kinase

signalling pathways, it is likely that in the brain it has the same function. Since mAKAP

forms such a large signalosome, it is currently unclear whether there is a single, large

complex that contains all of the mAKAP binding partners or, alternatively, whether there

are different mAKAP signalosomes that contain varying combinations of the signaling

molecules. We detected the highest mAKAP expression presynaptically in brain regions

such as the central amygdaloid nucleus, supraoptic nucleus, paraventricular hypothalamic

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General discussion

nucleus, and suprachiasmatic nucleus (Ostroveanu et al., 2009 submitted). Our finding that

mAKAP is abundantly expressed, particularly presynaptically, suggests an important role

for mAKAP in coordinating signal transduction pathways involved in adequate brain

functioning. This presynaptic expression pattern overlaps remarkably well with the

expression pattern for corticotrophin releasing factor (CRF) (Pilcher & Joseph, 1984). The

paralleled distribution of mAKAP with the CRF system suggests that mAKAP may also be

involved in a variety of functions such as emotion and autonomic responses but also

memory processes. Furthermore, we found a dramatic decrease in mAKAP expression in

all brain regions of aged mice. It has been shown that the expression levels of many genes

related to neuronal signaling, plasticity, and structure are changed in the hypothalamus and

cortex of the aged mouse brain (Jiang et al., 2001). Several proteins are downregulated in

the aged brain. For example, the level of PKC β1 is significantly decreased in aged rats as

assessed by RT-PCR and Western blotting (Yao et al, 1998). Moreover, in brain tissue from

elderly patients diagnosed with mild cognitive impairments several synaptic proteins (e.g.

synaptophysin) in the frontal and temporal cortex were shown to be downregulated (Counts

et al., 2006). Although it is difficult to speculate on the role of mAKAP in the brain on the

basis of its distribution, it may very well be that mAKAP contributes to changes in brain

functioning in aged mice and that it plays a role in cognitive processes.

A growing body of work on the role of AKAPs in the brain and other tissues provides

evidence that these multifunctional scaffolding proteins facilitate the fidelity of cAMP

signaling. This tight control of cAMP signaling is clearly important in the maintenance of a

healthy state, whereas the loss of this regulation might initiate diseases. How mAKAP

mediates the crosstalk and the integration of different signaling pathways via its

multimolecular protein complexes and what function it serves in the brain remains,

however, to be determined.

5. AKAPs as new therapeutic targets

Several studies have implicated the AKAP signalosome in ion channel modulation, G-

protein-coupled receptor desensitization, vesicular secretion, actin cytoskeletal dynamics,

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cell division and gene transcription regulated by cAMP, Ca2+, and lipid second messengers

(Michel & Scott, 2002). Since many of these cellular processes are altered in human

disease, understanding the mechanisms of AKAP signaling pathways may aid in the

development of novel therapeutics. So far traditional treatment in cognitive disorders has

focused on the manipulation of cell surface protein function. However, pharmacological

manipulation of cAMP signaling cascades in space and time may ultimately give us more

control on where and when events occur within the cell. Through careful intervention in the

coordination of signaling cascades we can gently nudge a cell into a certain direction. By

targeting anchoring proteins, like AKAPs, we could achieve this subtle intervention.

6. The role of Epac in learning and memory

Fairly recently, a novel PKA-independent cAMP effector was discovered and named

exchange protein directly activated by cAMP (Epac). In independent studies, two variants

of the Epac protein, Epac1 (also called cAMP-GEF-I) and Epac2 (also called cAMP-GEF-

II), were characterized (de Rooij et al., 1998; Kawasaki et al., 1998). Both Epacs function

as guanine nucleotide-exchange factors (GEFs) that specifically activate Rap GTPases

(Rap1 and Rap2) upon binding to cAMP. Although Epac proteins have been found to

control key cellular processes, including cellular calcium handling, integrin-mediated cell

adhesion, gene expression, cardiac hypertrophy, inflammation, and exocytosis (Pereira et

al., 2007; Oestreich et al., 2007; Hucho et al., 2005; Rangarajan et al., 2003; Lotfi et al.,

2006; Kang et al., 2003; Morel et al., 2005), their role in the brain just started to be

investigated. Since cAMP signaling is established to be of critical importance in learning

and memory, a potential role for PKA-independent cAMP signaling through Epac proteins

in the process of learning and memory may be expected. Recently, Epac has been linked to

synaptic transmission and neuronal excitability (Zhong & Zucker, 2005), was shown to

modulate membrane potentials in cultured cerebellar neurons, thereby potentially regulating

cellular excitability (Ster et al., 2007), and to enhance neurotransmitter release at excitatory

synapses (Gekel & Neher, 2008). Very recently it has been shown that Epac is involved in

both LTP (Gelinas et al, 2008) and LTD (Ster et al., 2009) in the mouse hippocampus.

156

General discussion

Since both changes in LTP and LTD that affect synaptic transmission in the hippocampus

are thought to be involved in memory formation, these data suggest furthermore the

involvement of Epac in learning and memory processes.

We showed that intrahippocampal delivery of 8-pCPT-2’O-Me-cAMP, a compound that

specifically activates Epac but not PKA, improves fear memory retrieval in contextual fear

conditioning whereas acquisition and consolidation were not affected (Ostroveanu et al.,

2009 submitted). The retrieval enhancing effect of the Epac activator was even more

prominent in the passive avoidance test, a behavioral paradigm based on associative

emotional learning similar to fear conditioning. Since the performance of the mice in the

retention tests may be influenced by the level of anxiety the animal experiences, we tested

the effect of 8-pCPT-2’O-Me-cAMP on anxiety behavior in an elevated plus maze.

Intrahippocampal 8-pCPT-2’O-Me-cAMP injection before the elevated plus maze had no

effect on anxiety. Therefore, the effect of Epac activation in the fear-motivated learning

tasks can be solely ascribed to enhanced memory retrieval of the association between the

electric footshock and the context (Ostroveanu et al., 2009 submitted). Ouyang and

colleagues also recently reported a role for Epac signaling in memory retrieval (Ouyang et

al., 2008). In their study, the memory retrieval impairment observed in dopamine-beta-

hydroxylase deficient mice could be rescued by intrahippocampal (i.h.) injection of a

selective PKA activator together with a selective Epac activator, whereas injection of one

of the activators alone did not overcome the retrieval deficit (Ouyang et al., 2008).

Interestingly, when cAMP agonists were infused into the dorsal hippocampus of wild-type

mice 30 min before testing retrieval, no significant effects on retrieval were observed when

the agonists were infused separately or in combination (Ouyang et al., 2008). The lack of

any effect on memory retrieval in the wild type mice is in contradiction with our findings

and may depend on the time of agonist delivery. Ouyang and colleagues performed the

injections 30 minutes before the retrieval, whereas in our experiments, Epac agonist was

injected into the CA1 are of the hippocampus 10 minutes before the memory test.

It has been shown that 8-pCPT-2’O-Me-cAMP activates both Epac1 and Epac2. Therefore,

the enhanced memory retrieval effect cannot be attributed to a particular Epac. We showed

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Chapter 7

that Epac2 is abundantly expressed throughout the whole mouse brain, whereas only low

levels of Epac1 were detected. Using semi-quantitative RT-PCR for Epac1 and Epac2 with

mRNA isolated from the hippocampus, Epac2 mRNA could be detected much earlier as

Epac1 mRNA, pointing towards much higher Epac2 than Epac1 mRNA levels in the

hippocampus (Ostroveanu et al., 2009 submitted). Our data are consistent with a previous

study from Kawasaki et al. who also reported a high expression of Epac2 in the rat brain

whereas Epac1 was barely detectable (Kawasaki et al., 1998). To date, no Epac-specific

inhibitors are available. Therefore, we established a protocol for in vivo lipid mediated

siRNA gene silencing in the mouse brain to investigate the role of Epac2 in learning and

memory. Local Epac2 silencing in the CA1 area of the hippocampus led to impaired

memory retrieval 3 days after conditioning whereas Epac2 silencing during the retention

test 17 days after conditioning had no effect on memory retrieval indicating a time-limited

function of Epac2 signaling after conditioning. The finding that Epac2 is abundantly

expressed in the mouse brain together with the finding that Epac2 silencing impairs

memory retrieval, suggests an important role for Epac2 in learning and memory. In our

experiments we could not completely exclude a role for Epac1 in these processes. Both

specific-Epac inhibitors and/or transgenic or knock-out mice will provide further evidence

on the role of Epac1 and Epac2 in cognitive processes.

Interestingly, expression levels of both Epac1 and Epac2 were reported to be altered in the

frontal cortex and hippocampal regions of brains showing Alzheimer’s pathology (McPhee

et al., 2005). Alzheimer disease (AD) is a neurodegenerative disease characterized by the

formation of β-amyloid plaques and neurofibrillary tangles. It has been shown that

amyloid-beta protein dimers isolated directly from Alzheimer's brains impair synaptic

plasticity and memory (Shankar et al., 2008). Moreover, in AD, neural degradation occurs

primarily in neurons involved in memory storage and retrieval (Arshavsky, 2006). So far

the relationship between changes in Epac expression and Alzheimer’s pathology remain

unclear, but it could very well be that Epac plays a role in the cognitive decline such as

observed in AD. This is also supported by the finding that the Epac2 levels are decreased in

AD. We have shown in our studies that activation of Epac in the mouse hippocampus

significantly and specifically improves the retrieval of fear memory. Considering both the

158

General discussion

lack and the need of drugs proven to be effective in improving memory retrieval, the

specific facilitating effect of Epac activation on retrieval we observed, is of particular

interest and deserves further research into the role of Epac signaling in cognitive processes

under physiological and pathological conditions.

Recent findings in several physiological systems acknowledged Epac as coordinator of

specific cAMP signals independent of PKA. Interestingly, it appears that in part, signals

carried through Epac are spatially and temporally compartmentalized. For example, in the

heart mAKAP is able to target Epac1 (Dodge-Kafka et al., 2005), while in the brain

AKAP150 binds both Epac1 and Epac2 (Nijholt et al, 2008). Thus, the spatio-temporal

integration of cAMP pathways via Epac, PKA, AKAPs and PDEs dramatically increases

the complexity and, consequently, the possible readouts of cAMP signalling. Moreover,

since both Epac and PKA are ubiquitously expressed, an increase in cAMP levels will lead

to the activation of both effectors which in turn can execute independent responses or

develop a cross-talk effect. For example, it has been reported that PKA and Epac can exert

opposing effects on the regulation of the PKB/AKT pathway. While PKA suppresses PKB

phosphorylation and activity, activation of Epac leads to increased PKB phosphorylation

(Mei et al., 2002; Nijholt et al., 2008). Interestingly, we recently showed that PKA and

Epac-mediated PKB/Akt phosphorylation is coordinated in neurons by AKAP150 (Nijholt

et al., 2008). In addition to the opposing effects, PKA and Epac may also work

synergistically in other systems. For example both the stimulation of neurotensin (Li et al.,

2007) and the attenuation of cAMP signaling through phosphodiesterases depend on the

activation of Epac and PKA (Dodge-Kafka et al., 2005).

Future research will lead to a better understanding of the physiological roles of both Epac

isoforms. Eventually, this may lead to a more detailed knowledge of the distinct role that

individual Epacs play in shaping cAMP signalling in the mammalian brain. This in turn

could result in the development of new strategies for selective pharmacologic manipulation

of the different cAMP signaling systems in cognitive processes under physiological and

pathological conditions.

159

Chapter 7

7. Overall conclusion and future perspectives

In the current thesis we aimed to investigate the role of two recently discovered, cAMP

signaling avenues, via A-kinase anchoring proteins (AKAPs) and via exchange protein

activated by cAMP (Epac), in memory processes. A large number of publications

established an instrumental role of cAMP signaling in learning and memory processes.

With the discovery of both AKAPs and Epac, cAMP research has undergone a revival and

there grew a general awareness that cAMP signaling is much more complex than was

initially believed. Accordingly, it has become apparent that AKAPs and Epac might

mediate a more fine-tuned level of organization for cAMP second messenger systems. Our

results yield important insights in the role of AKAPs, compartmentalized cAMP signaling

and Epac in the different stages of learning and memory.

It was previously believed that anchoring proteins in the brain were only scaffolds for the

signaling enzymes involved in cognitive processes. Nevertheless, recent data, including our

own, show a completely new role for AKAPs as dynamic and active contributors to the

molecular machinery of learning and memory. With the increased number of anchoring

proteins discovered in the brain, careful analysis on the individual role and contribution of

AKAPs in learning and memory will continue to be an important part of future research.

Unquestionably, further analysis of AKAP macromolecular complexes role in learning and

memory will be forthcoming from genetic approaches involving transgenic mice.

Genetically modified mice expressing truncated AKAP molecules that hinder tethering of

signaling enzymes, or mice lacking a particular AKAP, will permit a more precise

dissection of particular AKAP-PKA-signaling enzymes/complexes in vivo. These mice will

not only provide insight into the mechanisms of AKAP functions but may yield genetic

models of specific diseases.

In this thesis, we could also show that cAMP signals independent of PKA pathways are

essential events in learning and memory processes. Consequently, compelling evidence

points towards an important role for Epac in these processes. However, the mechanism by

160

General discussion

which Epac activation affects learning and memory is still unknown. Understanding how

exactly cAMP signals are coordinated will bring light on the elusive process of learning and

memory and memory retrieval in particular.

cAMP signaling was shown to be crucial in cognitive deficits and may consequently be a

promising target for therapeutic agents. Clearly, the ultimate goal of research on learning

and memory is translating knowledge from the laboratory to clinical applications. Thus, in

addition to the fundamental significance of understanding cAMP signaling cascades, our

research opens up new routes for the development of new therapeutic strategies for

memory-associated disorders and pathologies.

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Submitted

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