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The role of Akt in AMPA receptor insertion and LTP Yan, Yi 2007

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T H E R O L E OF Akt IN A M P A R E C E P T O R I N S E R T I O N A N D L T P by Y I Y A N B . S c , Peking University, 2003 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E S T U D I E S (Neuroscience) T H E U N I V E R S I T Y OF BRIT ISH C O L U M B I A January 2007 © Y i Yan , 2007 A B S T R A C T It has been widely accepted that long-term potentiation (LTP) in the hippocampal C A 1 region mostly results from increased insertion of post-synaptic a-amino-^ r 3-hydroxy-5-methyl-isoxazole-4-propionic acid receptors (AMPARs) . The previous study in our lab has shown that activation of phosphatidylinositol 3-kinase (PI3K) by selective stimulation of synaptic N-methyl-D-aspartate receptors ( N M D A R s ) is required for the increased cell surface expression of A M P A R s and the consequent LTP. However, the following signaling pathways still remain unknown. In the present study, the involvement of Akt , the primary downstream protein kinase of PI3K, was examined with a combination of electrophysiological, biochemical and molecular biological techniques. The study found that Ak t is required for the post-synaptic A M P A R insertion and LTP. Furthermore, the threonine 840 (Thr840) on G l u R l C-tail was identified as a novel Akt phosphorylation site, suggesting a potential mechanism by which Akt contributes to A M P A R incorporation and LTP. i i T A B L E OF C O N T E N T S Abstract „ . . . . i i Table of Contents i i i List of Figures v Acknowledgement v i i Introduction 1 Overview of the present study 1 Background and current knowledge 2 Long-term potentiation 2 Molecular structure of A M P A receptors 16 Ak t 17 Methods and materials 27 Cloning, expression and purification of G S T fusion proteins 27 In vitro phosphorylation assay with hippocampal slices 27 Slice preparation and recordings 29 In vitro Ak t kinase assay with G S T fusion proteins 30 Data analysis 31 Results 32 Akt activation during LTP in the C A 1 region of hippocampal slices by measuring Ser473 phosphorylation 32 The requirement of Akt for LTP in the C A 1 region of hippocampal slices i i i tested with the Akt dominate negative peptide and neutralizing antibody. 33 Akt phosphorylation of G l u R l C T on Thr840 36 G l u R l C T Thr840 phosphorylation during LTP in the hippocampal C A 1 region 40 LTP in the hippocampal C A 1 region was not blocked by G l u R l C T peptides 46 Discussion 48 The role of Akt in regulating synaptic plasticity 48 The role of G l u R l phosphorylation in A M P A R insertion and LTP 49 The mechanisms by which Akt contributes to LTP 51 Future direction 53 Significance of the present study 54 Bibliography 55 LIST OF F I G U R E S Figure 1 Protein-protein interaction of G l u R l and GluR2 with their binding proteins „ 10 Figure 2 Illustration of LTP induction, expression and maintenance „ 14 Figure 3 Illustration of the structure o f A M P A R subunit 17 Figure 4 Illustration of the structure o f Akt and its activation „ 20 Figure 5 Roles of Akt in regulating cell survival 23 Figure 6 Illustration of G l u R l C T mutants construction 28 Figure 7 Akt activation in the C A 1 region of hippocampus during LTP 34 Figure 8 Blockade of LTP in the C A 1 region of hippocampus by the Akt P H domain peptide (GST-Akt -AH) 35 Figure 9 Blockade of LTP in the C A 1 region of hippocampus by the Akt neutralizing antibody (Akt 1/2 N-19) 37 Figure 10 L T D in the C A 1 region of hippocampus was not blocked by the Akt neutralizing antibody (Akt 1/2 N-19) 38 Figure 11 In vitro Ak t phosphorylation of G l u R l C T but not GluR2 C T 39 Figure 12 In vitro Ak t phosphorylation of G l u R l C T on Thr840 41 Figure 13 In vitro neither P K A nor P K C phosphorylated G l u R l C T on Thr840 42 Figure 14 G l u R l Thr840 phosphorylation in the C A 1 region of hippocampus during LTP 44 Figure 15 Inhibition of G l u R l Thr840 phosphorylation in the C A 1 region v of hippocampus during LTP by A P V and P I3K inhibitor 45 Figure 16 LTP in the C A 1 region of hippocampus was not blocked by G l u R l C T (832-844) peptide 47 v i A C K N O W L E D G E M E N T There are many people I would like to thank for their assistance in the completion of this degree. First of al l , I am grateful for my supervisor Dr. Yutian Wang who provided me with the opportunity to work in his wonderful lab. I appreciate his patience, expertise and instruction. I would like to extend my gratitude to many others in our lab, especially to Jie, who offered me generous assistance. I would like to thank the members of my committee for their valuable advices in the completion of my thesis. I also would like to give my appreciation to the CIFfR Neuroscience Training Progam which provided funding for my research. Finally, I am deeply grateful for my parents and my girlfriend Jing for their love and support throughout the tenure of this degree. v i i I N T R O D U C T I O N I. Overview of the present study Long-term potentiation (LTP) represents as a long-term facilitation of synaptic transmission (Bliss and Gardner-Medwin, 1973; Abraham et al., 2002) which may play important roles in mediating various neuronal functions including learning and memory (Bliss and Collingridge, 1993; Lisman et al., 1997; Malenka and N ico l l , 1999). There are two major subfamilies of post-synaptic ionotropic glutamate receptors closely associated with LTP: the a-amino-3-hydroxyl-5-methyl-4-isoxazolepropionic acid receptor ( A M P A R ) and the 7V-methyl-D-aspartate receptor ( N M D A R ) (Hollmann and Heinemann, 1994). N M D A R activation is required for the induction of LTP, while A M P A R activity primarily expresses the plastic changes (Bear and Malenka, 1994; Bl iss and Collingridge, 1993; Malenka and N ico l l , 1999). It has been widely accepted that NMDAR-dependent LTP in hippocampal C A 1 region mainly results from post-synaptic A M P A R insertion to the synaptic membrane (Benke et al., 1998; Takahashi et al., 2003). However, the mechanisms underlying A M P A R insertion during LTP are quite controversial and not completely clear. It has been reported that many intracellular pathways, including calcium/calmodulin dependent kinase II (CaMKII) , protein kinase A ( P K A ) , protein kinase C (PKC) and mitogen-activated protein kinase ( M A P K ) are implicated in the A M P A R insertion during NMDAR-dependent LTP (Goelet et al., 1986; Alberini et a l , 1995; Zhu et a l , 2002; Boehm et al., 2006). Phosphoinositide 3-kinase (PI3K), a l ipid kinase, plays a controversial role in LTP induction (Opazo et al., 2003), expression 1 (Sanna et al., 2002) and maintenance (Raymond et al., 2002) in the hippocampal C A 1 region. A previous study in our laboratory has found that inhibition of P I3K prevents glycine-induced A M P A R insertion and LTP, while intracellular application of active PI3K potentiates mEPSCs and occludes glycine-induced LTP, indicating the requirement of P I3K for A M P A R insertion and LTP (Man et al., 2003). A s the primary downstream protein of PI3K, Akt has also been found as a novel modulator of synaptic plasticity. For example, our previous study has shown that Akt directly phosphorylates the G A B A A R p2 subunit on Ser410 and hence induces translocation of G A B A A R to the cell surface, which regulates the strength of G A B A A R transmission (Wang et al., 2003). According to the above studies, very possibly, Akt is also involved in regulation of A M P A R transmission. Thus, I hypothesize that Akt plays a critical role in NMDAR-dependent facilitation of A M P A R insertion and LTP in the hippcampal C A 1 region. To test this central hypothesis, the following aims wi l l be studied. 1) Investigate the activation of Ak t during LTP in the C A 1 region of hippocampus; 2) Test the requirement of Ak t for A M P A R insertion and LTP in the hippocampal C A 1 region; 3) Examine the mechanisms by which Akt contributes to A M P A R insertion and LTP in the C A 1 region of hippocampus. II. Background and current knowledge 1. Long-term potentiation 1). L T P and glutamate receptors Long-term potentiation, resulting from coincident pre- and post-synaptic activities, represents a facilitation of synaptic transmission that can last for hours, weeks or even 2 months (Bliss and Gardner-Medwin, 1973; Abraham et al., 2002). So far, LTP has been studied in different regions of central nervous system in different species (Fox, 2002; Ji et al., 2003; Nosten-Bertrand et a l , 1996; Urban et al., 1996). LTP as wel l as long-term depression (LTD) at the glutamatergic synapses of the hippocampal C A 1 region are considered as two important models of synaptic plasticity (Bliss and Collingridge, 1993; Malenka and N ico l l , 1999) which may have important roles in mediating various neuronal functions including learning and memory (Bliss and Collingridge, 1993; Lisman et al., 1997; Malenka and N ico l l , 1999). The mechanisms of LTP are now under hot investigation and most of the studies have been carried out in the hippocampal C A 1 region. Glutamate receptors, activated by glutamate released from excitatory synapses of the mammalian C N S , are greatly involved in LTP and LTD induction and expression. There are two major subfamilies of post-synaptic ionotropic glutamate receptors: A M P A R s and N M D A R s (Hollmann and Heinemann, 1994). A t the resting membrane potential, A M P A R s mediate most of the synaptic transmission, whereas N M D A R s are blocked by M g 2 + and therefore contribute little to basal excitatory post-synaptic current. When the post-synaptic membrane is depolarized, the M g blockade on N M D A R s is relieved and activation of N M D A R s initiates various forms of synaptic plasticity due to the high calcium permeability of N M D A R s (Bliss and Collingridge, 1993). Although the activation of N M D A R s is required for the induction of both LTP and L T D , changes in A M P A R activity accounts for the expression of the plasticity (Bear and Malenka, 1994; Bl iss and Collingridge, 1993; Malenka and N ico l l , 1999). How the A M P A R activity is changed is being widely investigated and l ikely involves both a pre-synaptic mechanism 3 by the alteration of pre-synaptic structure and glutamate release and a post-synaptic mechanism by long-term modification of post-synaptic A M P A R s (Bliss and Collingridge, 1993; Bolshakov et al., 1997; Bolshakov and Siegelbaum, 1994; Malenka and N ico l l , 1999). 2). LTP induction The induction of LTP requires coincident pre- and post-synaptic activities. Two kinds of protocols are often used to induce LTP in hippocampal C A 1 region. One is to apply high frequency stimulation (HFS) to Schaffer collateral/commissural fibers projecting from C A 3 to C A 1 pyramidal neurons, and the other is to simultaneously pair 2 H z electric stimuli with a depolarization of the post-synaptic neuron (Gustafsson et al., 1987; Markram et al., 1997; B i and Poo, 1998). A t glutamatergic synapses in the C N S , N M D A R s are the initiators for post-synaptic change during LTP (Collingridge et al., 1983). Glutamate is released from the pre-synaptic site into the synaptic cleft when an action potential is delivered to the pre-synaptic terminal. However, the binding of glutamate to post-synaptic N M D A receptors cannot open the N M D A R ion channels due to the M g 2 + blockade at the resting membrane potential (Nowak et al., 1984). A n influx of N a + and C a 2 + through N M D A R s occurs when this M g 2 + blockade is removed by sufficient post-synaptic depolarization. This C a 2 + influx has been widely accepted as the trigger of LTP induction (Lynch et al., 1983; Malenka et al., 1988). Many calcium downstream signaling pathways, such as C a M K I I or cAMP-dependent pathway, are involved in the LTP initiation. The activation of protein kinase, like C a M K I I and P K A , results in receptor phosphorylation and change of their intrinsic properties or gene 4 expression alteration via transcription factors (Goelet et al., 1986; Alber ini et al., 1995). The CaMKII-dependent insertion of A M P A R s might also involve activation of the Ras p42/44 M A P K pathway (Zhu et al., 2002). In addition, P K C (Boehm et al., 2006) and PI3K (Man et al., 2003) have also been reported to be implicated in the induction of NMDAR-dependent LTP. However, the molecular mechanisms of LTP induction may be different from synapse to synapse. LTP induction is NMDAR-dependent and mainly due to the post-synaptic mechanism (McNaughton, 1982; Manabe et al., 1992; N ico l l and Malenka, 1995) at both Schaffer co l la tera l -CAl pyramidal neuron synapses (Collingridge et al., 1983) and medial perforant path-dentate gyrus granule cell (Morris et al., 1986; Errington et al., 1987), whereas the LTP induction at the mossy f iber -CA3 pyramidal cell synapse in hippocampus is NMDAR-independent and relies on a pre-synaptic mechanism (Harris and Cotman, 1986; Weisskopf and N ico l l , 1995). In addition, these two sets of synapses employ different signaling mechanisms. For example, C a M K I I signaling is required for the former but not the latter (Zhang et al., 2005; Cooke et al., 2004). 3). LTP expression Although LTP expression may result from both pre- and post-synaptic mechanisms (Kauer et al., 1988; Malgarol i and Tsien, 1992), people have recently paid more attention to the latter. Many works have been focused on changes in the intrinsic conductance of glutamate receptor channels (Derkach et al., 1999; Lee et al., 2003) or in the number of glutamate receptors inserted into the synaptic membrane (Takahashi et al., 2003). The mechanisms are still controversial and none is completely characterized. 5 It has been widely accepted that NMDAR-dependent LTP in hippocampal C A 1 region mainly results from post-synaptic A M P A R insertion to the synaptic membrane in a membrane fusion manner. Once the postsynaptic membrane fusion is blocked, both the recombinant homomeric G l u R l A M P A R s insertion (Hayashi et al., 2000; Shi et al., 1999) and hippocampal LTP is inhibited (Lledo et al., 1998). Constitutive insertion of native A M P A R s into synaptic plasma membranes of cultured hippocampal neurons have been visualized and quantified (Lu et al., 2001), providing the first evidence that facilitated membrane fusion-dependent A M P A R insertion into post-synaptic membranes give rise to the expression of LTP in hippocampal cultures. These results have been supported by studies from several other labs (Liao et al., 2001; Passafaro et al., 2001). Changes of A M P A R s contribute to LTP expression in two ways: 1) functional alteration of existing A M P A R s at the synapse; 2) the rapid recruitment of new A M P A R s to synapse (Benke et al., 1998). The latter mechanism has been widely investigated and a concept of "silent synapse" which expresses only N M D A R s but not A M P A R s stood out (Isaac, 2003; Voronin and Cherubini, 2004). LTP is believed to involve the "unsilencing" of silent synapses by rapid insertion of A M P A R s to the plasma membrane of synapses that previously lack A M P A R s . With application of pH-sensitive G F P - A M P A R constructs (Ashby et al., 2004) or antibodies that recognize extracellular epitopes of native A M P A R subunits (Richmond et al., 1996), people could visualize A M P A R s in l iving neurons to investigate how their movement relates to synaptic plasticity (Collingridge et al., 2004). It has been reported that H F S induces GluRl-containing receptors insertion to cell surface through a mechanism of P K A and CaMKII-mediated phosphorylation (Hayashi 6 et al., 2000; Esteban et al., 2003), and after H F S , constitutive recycling process occurs, in which G luR2- or GluR3-containing receptors follow to exocytose and replace GluRl-containing receptors (Shi et al., 2001). In addition to incorporation into the plasma membrane, the number of A M P A R s can also be regulated by lateral diffusion in the plasma membrane during synaptic plasticity. Measurement by single molecule fluorescence microscopy has revealed that spines contain both immobile and mobile A M P A R s , and stimulation with glutamate increases the rate of diffusion of synaptic A M P A R s , decreases the proportion of immobile receptors and increases the proportion of receptors in the area around synapses (Tardin et al., 2003). Thus, both insertion of A M P A R s into the plasma membrane and a lateral movement of A M P A R s into the synapse contribute to LTP expression. Consistent with this idea, phosphorylation o f G l u R l C-terminal (CT) on Ser845 has been shown to drive A M P A R s to extra-synaptic sites for subsequent lateral delivery to synapses during LTP (Oh et al., 2006). Recently, the mechanisms, by which A M P A R s incorporate into synapses during LTP, have been widely debated. It is thought that G l u R l subunits are important for A M P A R insertion during LTP while the GluR2 subunits are important for constitutive cycling o f A M P A R s and L T D (Shi et al., 1999; Hayashi et al., 2000; Shi et al., 2001). It has been demonstrated that G l u R l C T plays an obligatory role in A M P A R insertion in both cultured neurons (Passafaro et al., 2001) and cultured hippocampal slices (Hayashi et al., 2000), since it contains many potential phosphorylation sites and protein-protein interaction regions. Several groups have identified G l u R l C T Ser831 as a CaMKI I or P K C phosphorylation site (Barria et a l , 1997; Mammen et al., 1997; Soderling and 7 Derkach,. 2000), Ser845 as a P K A phosphorylation site (Roche et al., 1996) and Ser818 as a P K C phosphorylation site (Boehm et al., 2006), all of which are involved in A M P A R insertion and LTP expression. In addition to the phosphorylation mechanism, A M P A R insertion is also regulated by proteins which directly interact with A M P A R subunits. Although some studies have found that deletion of the last seven residues of G l u R l C T that comprise the P D Z ligand does not affect basal G l u R l synaptic localization, basal synaptic transmission, long-term potentiation or long-term depression (K im et al., 2005), this P D Z ligand is still considered to be important for the expression of C A 1 hippocampal synaptic plasticity since many proteins bind to it. The PDZ-domain-containing synapse-associated protein 97 (SAP97) is known to bind with G l u R l when phosphorylated by C a M K I I and form a complex with G l u R l and myosin-VI, which drives G l u R l into spines (Mauceri et al., 2004; Wu et al., 2002). This also indicates the involvement of this motor protein in A M P A R trafficking. It is also found that over-expression of post-synaptic density protein 95 (PSD-95) facilitates AMPAR-mediated synaptic transmission and occludes LTP (Stein et al., 2003; Ehrl ich and Malinow, 2004), and an interaction of PSD-95 with A M P A R s through stargazin is believed to be involved in this process (Chen et al., 2000). Transmembrane A M P A R regulatory proteins (TARPs) are known to strongly associate with A M P A R s throughout the trafficking pathway (Bredt and N ico l l , 2003; Tomita et al., 2004). In addition to the G l u R l subunit, the GluR2 subunit also plays a role in A M P A R trafficking by interacting with many binding proteins. For example, N-ethylmaleimide sensitive factor (NSF), an ATPase, has been first identified as a direct binding protein of 8 GluR2 subunit involved in membrane fusion events (Nishimune et al., 1998; Osten et al., 1998), and both the number of surface A M P A R s and evoked AMPAR-mediated synaptic transmission are reduced when the G l u R 2 - N S F interaction is interrupted (Nishimune et al., 1998; Noel et al., 1999; Luscher et al., 1999). Another region of the GluR2 C T interacts with the P D Z domain-containing proteins such as glutamate receptor-interacting protein (GRIP), A M P A R binding protein (ABP) and protein interacting with C-kinase (PICK1). These proteins have multiple functions such as anchoring A M P A R s at both synaptic (Dong et al., 1997; Osten et al., 2000) and intracellular (Daw et al., 2000) locations, releasing A M P A R s from intracellular membranes (Daw et al., 2000) or localizing other proteins close to A M P A R s . Furthermore, GRIP also binds to GRIP associated protein 1 ( G R A S P 1), l inking A M P A R s to Ras signaling (Ye et al., 2000) which is known to be implicated in A M P A R insertion during LTP (Zhu et al., 2002). Thus, protein-protein interaction of A M P A R subunits with their binding proteins provides an important way for regulating the A M P A R insertion. F ig . 1 shows the protein-protein interaction of G l u R l and GluR2 with their binding proteins. 4). LTP maintenance Many protein kinases are known to be involved in LTP maintenance. It has been reported that the treatment with N M D A or glutamate produces an increase in CaMKI I and 42-kDa M A P K activity in cultured hippocampal neurons (Fukunaga et al., 1993) and the inhibitor of M A P K blocks LTP maintenance (Rosenblum et a l , 2000; Rosenblum et al., 2002; Waltereit and Weller, 2003; Sweatt, 2004). The increase of C a M K T V activity is also observed during LTP, and this activation of C a M K T V happens in the nuclei of C A 1 9 Figure 1. Protein-protein interaction of G l u R l and GluR2 with their binding proteins. SAP97: synapse-associated protein 97; T A R P : transmembrane A M P A R regulatory protein; PSD-95: post-synaptic density protein 95; N S F : N-ethylmaleimide sensitive factor; P I C K 1 : protein interacting with C-kinase; GRIP: glutamate receptor-interacting protein; A B P : A M P A R binding protein. (Collingridge et al., 2004) 10 pyramidal neurons (Miyamoto, 2006). A n unusual, persistently active kinase—the brain-specific, atypical P K C isoform, protein kinase Mzeta ( P K M Q has received much attention, since it has been reported to be both necessary and sufficient for LTP maintenance (Sacktor et al., 1993; L ing et al., 2002; Sajikumar et al., 2005; Serrano et al., 2005; L ing et al., 2006). PKMC, strongly potentiates AMPAR-mediated E P S C s when applied into C A 1 pyramidal cells in hippocampal slices (Ling et al., 2002; L ing et al., 2006), and this potentiation is inhibited by bath application of P K M ^ inhibitory peptide (ZIP) (Serrano et al., 2005). Inhibition of PKM<; by ZIP reverses established late LTP but has no effect on early LTP or basal synaptic transmission (Ling et al., 2002; Serrano et al., 2005, Sajikumar et al., 2005). A very recent study has shown that ZIP, injected into the rat hippocampus, both reverses LTP maintenance in vivo and produces persistent loss of 1-day-old spatial information (Pastalkova et al., 2006). However, activity of synaptic kinases is not a sufficient mechanism for LTP maintenance over the long term (Chen et al., 2001). Protein phosphorylation may act as a trigger for new gene transcription and protein synthesis which occur during the later phase of LTP (Krug et al., 1984; Nguyen et a l , 1994). But, how do the signals transfer to the nucleus to induce gene expression to sustain LTP? One well-characterized pathway is the cAMP-dependent kinase signaling pathway which regulates gene expression through direct phosphorylation and activation of c A M P response element-binding protein ( C R E B ) , a constitutively expressed transcription factor. In addition, C R E B can be phosphorylated directly or indirectly by several other protein kinases (Shaywitz and Greenberg 1999). For example, C a M K T V was the major C R E B kinase in the response to 11 neuronal stimulation in cultured rat hippocampal neurons (Kasahara et al., 2000; Kasahara et a l , 1999), and C a M K T V and M A P K pathways act as C R E B kinases in the early and late phases of LTP, respectively (Miyamoto, 2006). Indeed, C R E B phosphorylation is persistently increased following LTP stimulation (Miyamoto, 2006) and LTP is known to induce C R E B phosphorylation and cAMP-dependent response element (CRE)-mediated gene expression (Impey et al., 1996; Schulz et al., 1999). C R E B once phosphorylated, forms homodimers or heterodimers with other members of the B-Zip family of transcription factors (Herdegen and Leah 1998), and the complex then binds to the C R E in the promoter region of responsive genes, thereby stimulating gene expression. The expression of the c-Fos protein is significantly increased during the late phase of LTP suggesting that phosphorylated C R E B stimulates the expression of c-Fos (Miyamoto, 2006). In addition, other constitutive transcription factors, such as E lk -1 , are also activated by H F S (Davis et al., 2000), indicating a cross-talk between different pathways. One of the results of neuronal gene transcription and protein synthesis could be synaptic structural remodeling which may be an important mechanism underlying LTP maintenance. Indeed, many studies have found the structure alteration during LTP. For example, rapid changes in length of spines have been observed following LTP induction (Yuste and Bonhoeffer, 2001). A number of studies have reported increases in synaptic contact area or density for certain subtypes of axospinous synapses up to 24 hours after LTP induction (Desmond and Levy, 1986; Stewart et al., 2000; Toni et al., 1999), and in one study, an increase in synaptic contact area remains observable 5 days after LTP 12 (Weeks et al., 2001). However, whether such a structural change is sufficient to maintain LTP has not been explicitly addressed. New techniques have been developed in order to solve this problem (Toni et al., 1999; Ostroff et al., 2002; Fukazawa et a l , 2003). It has been proposed that newly formed functional synapses participate in LTP maintenance (Toni et al., 1999). However, it is considered that other changes rather than formation of new synapses may contribute more significantly to LTP persistence (Fiala et al., 2002). One alternative is that the structural modification occurs on existing synapses to sustain LTP. To support this, an enlargement of existing synapses (Desmond and Levy, 1986; Stewart et al., 2000) and an increase in the length of synaptic oppositions (Fukazawa et al., 2003) have been found associated with LTP persistence. The cytoskeleton system, like actin, is greatly involved in these synaptic structural changes (Shi et al., 2001, Fukazawa et al., 2003). LTP-associated enlargements of synaptic zones also involves movement of polyribosomes from dendrites into spines (Ostroff et al., 2002), indicating a role of protein synthesis in the synaptic structural modifications. Fig.2 shows the illustration of LTP induction, expression and maintenance. 5). LTP and learning and memory Three characteristics make LTP as a potential candidate mechanism for learning and memory: longevity, input-specificity (Andersen et al., 1980; Barrionuevo and Brown, 1983) and associativity (McNaughton et a l , 1978; Levy and Steward, 1979). Considerable studies have provided evidences that LTP and learning and memory share similar molecular mechanisms. When N M D A R s are blocked by antagonists such as A P V , impairment of learning in many kinds of hippocampus-dependent memory tasks happens 13 Q ^ Q pre-synaptic I L-Glu NMDAR AMPAR G P C R - I H H I C a 2 t CaMKIV CaMKII PKC PI3K C R E B post-synaptic gene transcription and protein synthesis structural remodeling Figure 2. Illustration of LTP induction, expression and maintenance. L-Glu: L-glutamate; N M D A R : N M D A receptor; AMPAR: A M P A receptor; GPCR: G protein-coupled receptor; P K A : protein kinase A ; PKC: protein kinase C; CaMKII: calcium/calmodulin dependent kinase II; CaMKIV: calcium/calmodulin dependent kinase IV; M A P K : mitogen-activated protein kinase; PI3K: phosphoinositide-3 kinase; CREB: cAMP response element-binding protein. (Miyamoto, 2006) 14 (Morris et al., 1986; Abraham and Mason, 1988). Moreover, N R 1 conditional knockout mice, in which knockout of the N R 1 gene only occurs in the hippocampal C A 1 region, fail to exhibit LTP in C A 1 region and also show specific spatial learning and memory deficits (Tsien et al., 1996). These studies strongly suggest that NMDAR-dependent LTP play a role in hippocampus-dependent learning and memory. Some evidence indicates that LTP and memory share the same intracellular signaling pathways. For example, CaMKII-dependent and cAMP-dependent signaling pathways are involved in both LTP and learning and memory. Complete blockade of LTP induction in hippocampal C A 1 region is found in mice with a mutated aCaMKII (T286A), and so is the impairment of hippocampus-dependent learning and memory (Giese et al., 1998; Cooke et al., 2004). The cAMP-dependent signaling pathway sustains both LTP and long-term memory (Abel et al., 1997; Nguyen and Kandel, 1997), whereas a decrease of P K A activity by over-expression of a regulatory P K A subunit prevents both LTP and long-term memory (Abel et al., 1997). Over-expression of adenylyl cyclase, leading to a high concentration of c A M P and hence an increase of P K A activity, enhances both LTP and learning (Wang et al., 2004). Activation of M A P K , downstream protein kinase of P K A , is enhanced after hippocampus-dependent learning in mice, and the inhibitor of M A P K blocks both LTP maintenance (Rosenblum et al., 2000; Rosenblum et al., 2002; Waltereit and Weller, 2003; Sweatt, 2004) and long-term memory formation (Atkins et al., 1998; B lum et al., 1999; Bozon et al., 2003). C R E B is downstream of this P K A - M A P K pathway. Both mutant mice with a targeted disruption of C R E B and transgenic mice expressing a repressor of C R E B have deficits in sustainable LTP and hippocampus-dependent long-term memory 15 (Bourtchuladze et al., 1994; Bozon et al., 2003). Furthermore, mice expressing an inhibitor of an endogenous C R E B repressor have enhanced LTP and long-term memory (Chen et al., 2003). Recently, a study has shown that the P K M ^ inhibitory peptide, injected into the rat hippocampus, both reverses LTP maintenance in vivo and produces a persistent loss of 1-day-old spatial information, suggesting that the mechanism maintaining LTP sustains spatial memory (Pastalkova et al., 2006). Together, all the studies suggest a strong correlation between LTP and hippocampus-dependent learning and memory. Thus, LTP is currently considered as the favored candidate mechanism for learning and memory in the C N S . 2. Molecular structure of A M P A receptors A M P A R s are tetramers composed of four homologous subunits G l u R l - G l u R 4 (Wisden and Seeburg, 1993; Hollmann and Heinemann, 1994; Rosenmund et al., 1998). A M P A R s containing either G l u R l / G l u R 2 or GluR2/GluR3 subunits are known to be predominant in rat mature hippocampus (Wenthold et al., 1996). Complexes of G luR2/GluR4 are found in immature hippocampus and other mature brain areas (Zhu et al., 2000). The current topology model suggests that each A M P A R subunit comprises an extracellular N-terminal, an intracellular C-terminal, three transmembrane domains, M D 1 , M D 3 and M D 4 as wel l as a re-entrant membrane region corresponding to M D 2 (Fig. 3). The sequences of N-terminals and transmembrane domains of all the subunits are similar, whereas their C-terminals are different, either long or short. G l u R l and GluR4 mainly contain long C-tails, while GluR2 and GluR3 have short C-tails. A s we 16 , Ser831 Figure 3. Illustration of the structure of A M P A R subunit. M D : membrane domain. P K A : protein kinase A ; P K C : protein kinase C; CaMKI I : calcium/calmodulin dependent kinase II. (Bredt and N ico l l , 2003) 17 discussed above, the C-terminal is a very important region containing several phosphorylation sites for protein kinase and some protein-binding motifs for protein-protein interactions (Malinow and Malenka, 2002). CaMKI I , P K A and P K C phosphorylate G l u R l C T on Ser831, Ser845 and Ser818, respectively, and P K C can also phosphorylate G l u R l C T on Ser831 (Barria et al.,1997; Mammen et al., 1997; Roche et al., 1996; Boehm et al., 2006). The subunit-specificity of these G luR C-tails is highly associated with their different roles in mediating A M P A R trafficking and synaptic plasticity. 3. Akt 1). Molecular structure of Akt Akt , a serine/threonine kinase, is also called protein kinase B (PKB) . It is a member of the cAMP-dependent P K A / P K G / P K C ( A G C ) superfamily of protein kinases sharing structural homology within their catalytic domain and similar activation mechanism. In mammals, three isoforms of A k t / P K B , which are A k t l / P K B a , A k t 2 / P K B 0 and Ak t3 /PKBy , have been identified (Jones et al., 1991; Cheng et al., 1992; Brodbeck et al., 1999). A l l of the three A k t / P K B isoforms comprise a conserved domain structure: an N-terminal pleckstrin homology (PH) domain, a central kinase domain and a C-terminal regulatory domain. The major structural features of Akt are illustrated in Fig. 4A. The N-terminal P H domain, originally found in pleckstrin which is the major phosphorylation substrate for P K C in platelets (Tyers et al, 1998), binds with membrane l ipid products such as phosphatidylinositol (3,4,5)-trisphosphate (PIP3) (Coffer and Woodgett, 1991; 18 Jones et al., 1991; Bellacosa et al., 1991) and phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) (James et al., 1996; Freeh et al., 1997). The kinase catalytic domain of Akt , located in the central region of the molecule, shares a high degree of similarity with other A G C kinases such as P K A , P K C , p70S6K and p90RSK (Peterson and Schreiber, 1999). Another feature in this region is a conserved threonine residue (Thr308 in A k t l / P K B a ) whose phosphorylation partially activates Akt (Alessi et al, 1996). The C-terminal regulatory domain contains the F - X - X - F / Y - S / T - Y / F hydrophobic motif (where X is any amino acid) that is characteristic of the A G C kinase family (Peterson and Schreiber, 1999). In mammalian A k t / P K B isoforms, this motif is identical (FPQFSY) . 2). Activation of Akt Two phosphorylation sites, Thr308 in the kinase domain and Ser473 in the C-terminal regulatory domain (Fig. 4A) , are very important for Akt activation. Activation of A k t l by growth factors involves a PI3K- and P H domain-dependent membrane translocation step, followed by phosphorylation of these two sites. Phosphorylation of Thr308 partially activates Akt and phosphorylation of both sites induces its full activation (Fig. 4B). However, phosphorylation of Ser473 alone has little effect on Akt activity (Alessi et al., 1996). Akt , the downstream effector of PI3K, is activated by Class I A and Class IB PI3K which are activated by tyrosine kinase and G-protein-coupled receptors, respectively (Wymann et al., 2003). Fol lowing its recruitment to these receptors in the plasma membrane, P I3K is activated and converts PI(4,5)P2 to PIP3. PIP3 levels are tightly regulated by the action of phosphatases such as P T E N , which removes phosphate from the 3-OH position and converts PIP3 back to PI(4,5)P2 (Simpson and Parsons, 19 PH domain T308 Kinase domain S473 Regulatory domain B Plasma Membrane <rJ!ffEfffr5»-H Kinase domain |— activated Akt Regulatory domain Figure 4. Illustration of the structure of Akt and its activation. A , The molecular structure of Akt; B, The activation process of Akt. PDK1: phosphoinositide-dependent protein kinase 1; PI3K: phosphoinositide-3 kinase; PI(4,5)P2: phosphatidylinositol (4,5)-bisphosphate; PIP3: phosphatidylinositol (3,4,5)-trisphosphate. (Song et al., 2005) 20 2001). PIP3 binds to P H domain of Akt and then recruit it to the plasma membrane where Akt is phosphorylated on Thr308 and Ser473 (Andjelkovic et al., 1997). It has been clarified that Thr308 is phosphorylated by phosphoinositide-dependent protein kinase 1 (PDK1) (Stephens et al., 1998). However, the mechanism of Ser473 phosphorylation is quite controversial. Some evidence suggests that this site can be autophosphorylated (Toker and Newton, 2000). Some labs report that Ser473 is modified by a distinct kinase such as phosphoinositide-dependent protein kinase 2 (PDK2) (Hi l l et al., 2001). The integrin-linked kinase ( ILK) has also been thought to play a role in the Ser473 phosphorylation (Delcommenne et al., 1998; Persad et al., 2001). Furthermore, several recent reports have investigated the possible role of tyrosine phosphorylation in Ak t regulation (Conus et al., 2002; Jiang and Qiu, 2003). In addition, a recent study also provides strong evidence suggesting that C a M K I I can phosphorylate Akt on Thr308, thereby rendering it active (Okuno et al., 2000). This may be a mechanism by which activation of C a M K I I contributes to A M P A R insertion and LTP. In addition to PI3K, P K A has been reported to activate Akt in a PI3K-independent manner, although the mechanism is not fully clear (Fil ippa et al., 1999). cAMP-elevat ing agents such as forskolin, chlorophenylthio-cAMP, prostaglandin-El, and 8-bromo-cAMP are shown to activate Akt through P K A (Sable et al., 1997; Fi l ippa et al., 1999). The P H domain of Akt is not required for this activation and phosphorylation on Thr308 but not Ser473 is required for the PKA- induced activation. Moreover, it is also shown that Akt can be directly activated by C a M K in vitro (Perez-Garcia et a l , 2004). Other reports indicate that Akt is activated by cellular stress and heat shock through association with 21 Hsp27 (Konishi et al., 1997) and the P-adrenergic agonist, isoproterenol, can activate Akt in a wortmannin (PI3K inhibitor)-resistant manner (Moule et al., 1997). The significance of these findings remains to be determined. 3). Substrate regulation by Akt-dependent phosphorylation Many targets are regulated by Akt phosphorylation (Datta et al., 1999; Braz i l and Hemmings, 2001; Scheid and Woodgett, 2001), which can be classified into two general categories depending on the consequences of Akt phosphorylation for their function. 1) Akt-dependent phosphorylation either increases or decreases the activity o f the substrates. For example, Akt-dependent phosphorylation activates 6-phosphofructo-2 kinase (PFK2) and nitric oxide synthase (NOS) (Deprez et al., 1997, Dimmeler et al., 1999; Fulton et al., 1999; Michel l et al., 1999) and, in contrast, inhibits G S K 3 , M A P K , Raf-1, B-Raf, mixed lineage kinase-3 ( M L K 3 ) and apoptosis signal-regulating kinase 1 (ASK1) (Cross et al., 1995, Rommel et al., 1999; Zimmermann and Moel l ing, 1999; Guan et al., 2000; K i m et a l , 2001; Barthwal et al., 2003). 2) Ak t phosphorylation increases the affinity of the substrate for interaction with 14-3-3 proteins. 14-3-3 proteins are widely expressed and retain phosphorylated Ak t substrates in the cytoplasm by specifically binding phospho-serine/threonine-containing polypeptides (Franke and Cantley, 1997). 3). Regulation of cell survival by Akt The Akt signaling pathway is now recognized as one of the most critical pathways in regulating cell survival (Fig. 5). The activation of Akt pathway provides cells with a survival signal that allows them to withstand apoptotic stimuli (Yao and Cooper, 1995). In recent years, many reports elucidate the important roles of Akt signaling pathway in 22 Cell survival Figure 5. Roles of Akt in regulating cell survival. M d m : murine double minute 2; I K K : I K B kinase; N F - K B : nuclear factor-KB; G S K 3 : glycogen synthase kinase 3; C R E B : c A M P response element-binding protein. (Song et al., 2005) 23 cell survival in several types of cancers (Bao et al., 2004; Shi et al., 2004; Gupta et al., 2004; Wendel et al., 2004). Akt contributes to cell survival in the following three ways: 1) Akt directly regulates apoptosis. Critical regulators in this pathway are various members of the Bcl -2 family. B A D , a member of the Bcl -2 family of proteins that binds to Bcl -2 or B c l - X and inhibits their antiapoptotic potential, is phosphorylated on Serl36 by Akt, which promotes cell survival (del Peso et al., 1997; Datta et al., 1997). Caspase-9, acting as an initiator and an effector of apoptosis (Donepudi et al., 2002), has been reported to be phosphorylated on Ser 196 by Akt, resulting in attenuation of its activity (Cardone et al., 1998). 2) Akt regulates cell survival through transcription factors that are responsible for pro- as wel l as anti-apoptotic genes. For example, Akt has been shown to regulate I K B kinase ( IKK) activity in both direct and indirect manner (Kane et al., 1999) and hence regulate the activation of the transcription factor nuclear factor-KB ( N F - K B ) , a key regulator o f the immune response (Barkett et al., 1999; L i and Verma, 2000; Lauder et al., 2001). Through regulating murine double minute 2 (Mdm2) by direct phosphorylation (Mayo et al., 2001; Gottlieb et al., 2002), Akt promotes the inactivation or degradation of p53 and undermines the p53-mediated proapoptotic transcriptional response. C R E B transcription factor can be phosphorylated by Akt on Ser l33, resulting in an increase in transcriptional activation of C R E B and affinity of C R E B to its co-activator C R E (Du and Montminy, 1998). 3) Akt regulates cell survival by modulating cell metabolism. For example, it has been reported that glycogen synthase kinase 3 (GSK3) is inhibited when phosphorylated by Akt (Cross et al., 1995), and the inhibition of G S K 3 has been found to be protective against apoptosis in many circumstances (Pap and Cooper, 1998). 24 4). Role of P I3K-Akt pathway in mediating synaptic plasticity PI3K is a known mediator of plasma membrane protein insertion (De Cami l l i et al., 1996; Passafaro et al., 2001; Pessin et al., 1999) and internalization processes (Rapoport et al., 1997; Shpetner et al., 1996). It plays a controversial role in LTP induction (Opazo et al., 2003), expression (Sanna et al., 2002) and maintenance (Raymond et al., 2002) in the hippocampal C A 1 region. The study in our lab has found that inhibition of P I3K prevents glycine-induced A M P A R insertion and LTP and intracellular application of active P I3K potentiates mEPSCs and occludes glycine-induced LTP (Man et al., 2003). Moreover, we have also found that the p85 subunit of P I3K has a direct interaction with G luR C T (Man et al., 2003). Thus, PI3K is required for glycine-induced LTP. In addition, CaMKI I has been shown to facilitate A M P A R insertion by activating Ras (Zhu et al., 2002), and Ras is known to activate PI3K (Rodriguez-Viciana et al., 1996; Downward, 1997). A s the primary downstream protein of PI3K, Akt has been found to directly phosphorylate G A B A A R (32 subunit on Ser410 and hence induce translocation of G A B A A R to the cell surface, which regulating the number of G A B A A R at postsynaptic sites (Wang et al., 2003), suggesting, for the first time, a role of Ak t in modulating synaptic plasticity. In addition, the Akt-dependent potentiation o f N and L type voltage-gated calcium channels by insulin-like growth factor-1 (Blair and Marshall , 1997; Blair et al., 1999) and an involvement of Akt in T G F Pi regulation o f a calcium-activated potassium channels (Lhuil l ier and Dryer, 2002) have been found, indicating a role of Akt in the regulation of voltage-gated ion channels in addition to ligand-gated ion channels. Taken together, Akt plays important roles not only in controlling neuronal survival but 25 also in the regulation of both ligand-gated and voltage-gated ion channel functions. 26 M A T E R I A L S A N D M E T H O D S 1. Cloning, expression and purification of G S T fusion proteins G S T - G l u R l C T (a.a.808-889), GST-GluR2 C T (the last 50a.a.), G S T - A k t - A H and GST-Ak t -AH(R25C) were constructed by subcloning corresponding P C R fragments into p G E X 4T-1 vectors, respectively. The truncated G S T - G l u R l C T and mutants, M831 , M838, M839, M840 and M845 (Fig 6), were generated using P C R methods or the Quick-Change Site Directed Mutagenesis K i t (Stratagene). A l l of G S T fusion proteins were expressed in B L 21 Escherichia coli and purified from bacterial lysates according to the manufacturer's protocol (Amersham pharmacia biotech). 2. In vitro phosphorylation assay with hippocampal slices Hippocampus was dissociated from the brain of 18-21-day-old SD rats and sliced. To induce LTP, the high frequency stimulation (3 trains of 100 Hz) was used. To make sure that LTP was induced, field recording was carried out first. Hippocampal slice (CA1 region only) extracts were prepared in heated homogenization buffer (1% SDS, 100 °C). The total amount of protein was measured by Ultrospe 3000 UV/v is ib le spectrophotometer (Pharmacia Biotech). The lysate was then subjected to S D S - P A G E and transferred to P V D F membrane (Bio-Rad Laboratories, Hercules, C A ) . The membrane was blocked with 5% bovine serum albumin (BSA) (Fisher Bioreagents, Ottawa, ON) for 1 hour at room temperature (25 °C) and then incubated with primary antibodies overnight at 4°C. The anti-phospho-Akt(S473) antibody (1:1000) (Cell 27 M831 (S831 A) M845(S845A) -IEFCYKSRSESKRMKGFCLIPQQSINEAIRTSTLPRNSGAGASGGGGSGENGRWSQDFPKSMQSIPSMSHSSGMPLGATGL M838(T838A), M839(S839A) or M840(T840A) Figure 6. Illustration of GhiRl CT mutants construction Signaling Technology, Inc., Danvers, M A ) and anti-phospho-GluRl(T840) antibody (1:400) (Abeam Inc., Cambridge, M A ) were used to detect phosphorylated proteins, and the Akt 1/2 (N-19) antibody (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, C A ) and G l u R l C T antibody (1:2500) (self-produced in our lab) were used to detect total proteins. The next day, membrane was rinsed and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (1:4000) (GE Healthcare U K Limited, U K ) for 1 hour at room temperature and bound antibodies were then detected by E C L Detection Reagents (Amersham Biosciences, U K ) . Quantifications were done by chemoluminescence and densitometric scanning of the films. The optical density of protein bands was analyzed by using ImageJ 1.36b (Wayne Rasband, National Institutes of Health, U S A ) . The following antagonist and inhibitors were used in the experiments: A P V (50 uM) (Sigma-Aldrich Canada, Ltd, Oakvil le, ON) , R p - c A M P S (25 uM) (Biomol International, LP, Plymouth Meeting, PA), wortmannin (10 uM) (Upstate, Lake Placid, N Y ) and chelerythrine (10 uM) (Sigma-Aldrich Canada, Ltd, Oakvi l le, ON) . 3. Slice preparation and recordings Hippocampal slices were prepared from 18-21-day-old SD rats. Animals were decapitated after being anaesthetized with an intraperitoneal injection of urethane (25%). Brains were sliced with a vibratome (LeicaVTlOOOS, Germany). During the experiment, slices were superfused continuously under submerged conditions, with a perfusion solution containing (in mM) : N a C l , 125; KC1, 2.5; C a C l 2 , 2; M g C l 2 , 1; N a H 2 P 0 4 , 1.25; N a H C 0 3 , 26; glucose, 25. Bicucull ine (10 uM) (Tocris Bioscience, El l isvi l le, M O ) was 29 added routinely to block G A B A A receptor-mediated responses and solution was bubbled with Carbogen (Carbon dioxide/oxygen oxidizing mixture, 5% CO2 and 95% O2) (Praxair Canada Inc., Mississauga, ON). The recording pipette (resistance, 8-10 MCI) contained (in mM) : Cs-gluconate, 132.5; C s C l , 5; M g C l 2 , 2; H E P E S , 10; E G T A , 0.5; ATP(K) , 4, and was corrected to 290 mosm/L with sucrose and to p H 7.2 with C s O H . QX314 (5 mM) (Alomone Labs, Jerusalem, Israel) was added to block N a + channels. E P S C s were recorded using the whole-cell configuration of the patch-clamp technique under voltage-clamp conditions. Whole cell recording was carried out with an Axopatch ID amplifier (Axon instruments, Union City, C A ) . A stimulation of 2 H z for 100 s was applied through a bipolar stimulating electrode placed over Schaffer collateral fibers between 300 um and 500 um from the recorded cells which are depolarized to -5 m V (LTP protocol) or -40 m V (LTD protocol) during the stimulation. The following antibodies and peptides were used in the experiments: G S T - A k t - A H (100 u.g/ml), GST-Ak t -AH(R25C) (100 ug/ml) (Self-produced), Akt 1/2 (N-19) antibody (100 ug/ml) (Santa Cruz Biotechnology, Inc., Santa Cruz, C A ) , normal IgG (100 u.g/ml) (Upstate, Lake Placid, N Y ) , G l u R l C T (832-844) peptide (500 ug/ml), G l u R l C T (832-844, pT840) peptide (500 ug/ml) and the scrambled G l u R l C T (832-844) peptide (500 ug/ml) (PepMetric Technologies Inc., Vancouver, B C ) . 4. In vitro Ak t kinase assay with G S T fusion proteins G S T - G l u R l CT, GST-GluR2 C T and mutated G S T - G l u R l C T (M831, M838, M839, M840 and M845) were incubated with recombinant active A k t l / P K B a (30 fxg/ml) 30 (Upstate, Lake Placid, N Y ) in Akt reaction buffer (50 m M Tr is -HCl pH7.5, 10 m M M g C l 2 , 1 m M D T T and cold ATP) at 30°C for 1 hour. The Akt phosphorylated G S T - G l u R l proteins were subjected to S D S - P A G E and transferred to P V D F membrane (Bio-Rad Laboratories, Hercules, C A ) . The following steps are the same as described in the in vitro phosphorylation assay. Phosphorylated proteins were detected by primary antibodies including phospho-(SerVThr) Akt substrate antibody (1:500) (Cel l Signaling Technology, Inc., Danvers, M A ) , anti-phospho-GluRl(T840) antibody (1:400) (Abeam Inc., Cambridge, M A ) , anti-phospho-GluRl(S831) antibody (1:500) (Upstate, Lake Placid, N Y ) , anti-phospho-GluRl(S845) antibody (1:500) (Upstate, Lake Placid, N Y ) , and total proteins were detected by anti-GST antibody (1:1000) (Amersham Biosciences, U K ) . In the radioactive experiment, G S T - G l u R l CT, GST-GluR2 C T and mutated G S T - G l u R l C T (M831, M838, M839, M840 and M845) were incubated with recombinant active A k t l / P K B a (60 ug/ml) (Upstate, Lake Placid, N Y ) in the Ak t reaction buffer with y - 3 2 P ATP (Amersham pharmacia biotech), and the incorporation of P into the protein bands was detected by autoradiography. 5. Data Analysis Data obtained were normalized to control (set at 1.0) where appropriate, and expressed as Mean ± S.E. A N O V A was used for comparison among multiple groups, followed by the Holm-Sidak test for comparison between two groups. Statistical significance was defined as P < 0.05. 31 R E S U L T S 1. Akt activation during LTP in the C A 1 region of hippocampal slices by measuring Ser473 phosphorylation To demonstrate that Akt is involved in LTP, the first step was to find out whether Akt activation is enhanced during LTP. To induce LTP in the C A 1 region of acute hippocampal slices, the high frequency stimulation (HFS, 3 trains of 100 Hz) was applied to the Schaffer collateral fibers projecting to C A 1 pyramidal neurons. Slices were collected and homogenized in the homogenization buffer right after and 2, 5, 10, 20, 30 min after the stimulation, respectively. Slices without stimulation were used as control. Then the slice extracts were tested by western blot to measure Akt activation. A s we know, Ak t consists of a C-terminal regulatory domain, a central kinase domain and an N-terminal pleckstrin homology (PH) domain which specifically recognizes and binds PIP3, the primary product of P I3K (Alessi et al., 1996; Coffer et al., 1998; Downward, 1998; Datta et al., 1999; Cantley, 2002). It is believed that Akt is activated by sequential events: translocation of Akt to PrP3-enriched plasma membranes through the binding of PIP3 to the Akt P H domain, and subsequent phosphorylation at Thr308 and Ser473 by phosphoinositide-dependent kinases (Alessi et al., 1996; Blume-Jensen and Hunter, 2001). Akt is thought to be partially activated when Thr308 is phosphorylated and fully activated when Ser473 is also phosphorylated. A n antibody against Akt with phosphorylation on Ser473 (anti-phospho-Akt(S473) antibody) was used to detect activated Akt. Another Akt antibody (Akt 1/2 N-19) was used to detect the total Akt for 32 normalization. A s shown in F ig . 7A, an increased activation of Akt occurred during the first 2 min after the LTP stimuli and lasted for less than 5 min. F ig . 7B shows that Akt activation level right after LTP stimuli was significantly increased compared to control, whereas no significant difference was shown 30 min after LTP stimuli. Thus, a fast activation of Akt occurred during LTP induction. 2. The requirement of Ak t for LTP in the C A 1 region of hippocampal slices tested with the Akt dominate negative peptide and neutralizing antibody Whole-cell recordings of AMPAR-mediated E P S C s in C A 1 neurons of acute hippocampal slices were performed to investigate whether the activation of Ak t is a requirement for LTP. To induce LTP in the C A 1 region of acute hippocampal slices, we used a pre-synaptic stimulation (2 H z for 100 s) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -5 m V of C A 1 pyramidal neurons. A s mentioned before, the binding of PIP3 to the Akt P H domain recruits Akt to the plasma membrane, and is an important step in the activation of Akt. The post-synaptic injection of a well-characterized Akt dominant negative peptide: G S T - A k t - A H (100 ug/ml) was carried out to block Akt activation. G S T - A k t - A H contains the PJJP3 binding P H domain, but lacks the kinase catalytic domain. Therefore, when delivered into the postsynaptic cells, it w i l l competitively inhibit PIP3 activation of endogenous Akt. I used GST-Ak t -AH(R25C) (100 ug/ml) as control. GST-Ak t -AH(R25C) carries a single mutation that removes the ability of the P H domain to bind PIP3 (Watton and Downward, 1999). The results show that G S T - A k t - A H (n=5), but not GST-Akt -AH(R25C) (n=5), blocked LTP (Fig. 8). 33 Time after LTP stimuli (min) (fl <-• Control II 2 5 10 20 30 IB: phospho-Akt (S473) J—55 KD IB: Akt1/2 N-19 ]—55 KD B ~o Q) N " T O E o c o > -i—* o 03 -i—i < 2.5 2 1.5 1 0.5 0 Control Right after 30 min after LTP stimuli LTP stimuli Figure 7. Akt activation in C A 1 region of hippocampal slices during LTP. To induce LTP, the high frequency stimulation (HFS, 3 trains of 100 Hz) was applied to the Schaffer collateral fibers projecting to C A 1 pyramidal neurons. Slices without stimulation were used as control. Slice extracts were tested by western blot to measure Akt activation. A , A significant enhancement of Akt activation (phosphorylation on Ser473) occurred during the first 2 min after the LTP stimuli and lasted for no more than 5 min. B, normalized results showing mean activation of Akt as mean±S.E. (n=9, A N O V A F=3.795, P O . 0 5 ) . *P<0.05 indicates significant difference from control. "Right after LTP stimuli" means that slices were collected and homogenized immediately after LTP stimuli. "30 min after LTP st imuli" means that slices were collected and homogenized 30 min after LTP stimuli. 34 => 3 r Q. E ro O C O Q _ U l LY. < D_ < E 2.5 1.5 0.5 0 - • - 1 0 0 ug/ml GST-Akt-AH, n=5 - 0 1 0 0 Mg/ml GST-Akt-AH(R25C), n=5 —A— blank control, n=10 LTP stimuli 10 15 20 25 30 35 Time (min) B GST-Ak t -AH GST-Akt -AH(R25C) 1 blank control 2 2 1: baseline (0-5 min); 2: after L T P stimuli (30-35 min) 50 ms 50 pA Figure 8. Blockade of LTP in the CA1 region of hippocampus by the Akt PH domain peptide (GST-Akt-AH). Whole-cell recordings of AMPAR-mediated EPSCs in CA1 neurons of acute hippocampal slices were performed. To induce LTP in the CA1 region of acute hippocampal slices, we used a pre-synaptic stimulation (2 Hz for 100 s) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -5 mV of CA1 pyramidal neurons. A , GST-Akt-AH only contains the PH domain and competitively inhibits PIP3 activation of endogenous Akt when intracellularly delivered. GST-Akt-AH(R25C), which carries a single mutation that removes the ability of the PH domain to bind PIP3, was used as control and normal intracellular solution without any drugs was used as blank control. B, representative traces of AMPAR-mediated EPSCs. 35 However, one concern was that this peptide might also inhibit the binding of PIP3 to other proteins containing a P H domain. Thus, there was a possibility that the blockade of LTP might be due to the inhibition on the activation of those other proteins. For further confirmation, an Ak t neutralizing antibody, Ak t 1/2 (N-19) antibody (100 u.g/ml), was used. This Akt N-terminal antibody specifically binds to Akt PH-domain and blocks its binding with PIP3 without affecting other proteins (Hi l l et al., 1999). We tested the effects of this antibody on both LTP and LTD. L T D was induced by a pre-synaptic stimulation (1 H z for 5 min) paired with a post-synaptic depolarization to -40 mV. We found that LTP (Fig. 9) but not L T D (Fig. 10) was blocked. Thus, we concluded that Akt activation is required for LTP but not LTD . 3. Akt phosphorylation o f G l u R l C T on Thr840 Next, we investigated the mechanism by which Akt contributes to LTP. A s we know, Akt is a protein kinase and exerts its function primarily by phosphorylating its substrate proteins. Using an in vitro Ak t phosphorylation assay, we found that active Akt specifically phosphorylated G l u R l CT, but not GluR2 C T (Fig. 11 A ) , which was further confirmed by experiments with 3 2 P detection (Fig. 11B). We noticed that there is a candidate motif (NEArRTST 8 4oLPRNSGA) for Akt phosphorylation on G l u R l C T and Thr840 is the most l ikely phosphorylation site. To find out whether Thr840 is phosphorylated by Akt , we constructed several G l u R l C T mutants, M831(S831A), M838(T838A), M839(S839A), M840(T840A) and M845(S845A), by site-directed mutation (Fig. 6). In these mutants, the serines or threonines were mutated to alanine. 36 A dj B -o ° -«—' "5. o CO Q_ LU cr: < Q . 2 h 1.5 < 5 1 CD E o 0.5 L T P stimuli 10 - • - 1 0 0 Mg/ml Aktl/2 (N-19) antibody, n=5 -A-100 Mg/ml normal IgG, n=4 - A - blank control, n=5 20 30 40 Time (min) 50 60 Akt1/2 (N-19) antibody J rv Normal IgG Blank control 1: baseline (0-5 min); 2: after LTP stimuli (60-65 min) 50 ms Figure 9. Blockade of LTP in the CA1 region of hippocampus by the Akt neutralizing antibody (Aktl/2 N-19). Whole-cell recordings of AMPAR-mediated EPSCs in CA1 neurons of acute hippocampal slices were performed. To induce LTP in the CA1 region of acute hippocampal slices, a pre-synaptic stimulation (2 Hz for 100 s) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -5 mV of CA1 pyramidal neurons was used. A, Aktl/2 (N-19), when intracellularly applied, binds with Akt and blocks its function to phosphorylate its substrates. Normal IgG was used as control and normal intracellular solution without any drugs was used as blank control. B , representative traces of AMPAR-mediated EPSCs. 37 "O -*—* E 03 O CO D_ LU < Q_ 1.2 0.8 0.6 a! 0.4 N 03 E i_ o 0.2 0 - • - 1 0 0 ug/ml Akt1/2 (N-19) antibody, n=9 -A-100 ug/ml normal IgG, n=6 - A - blank control, n=6 LTD stimuli. 10 20 30 40 Time (min) 50 60 B Akt l /2 (N-19) antibody 2 " = = = ~ 1: baseline (0-5 min); 2: after LTP stimuli (65-70 min) 50 pA 50 ms Figure 10. L T D in the C A 1 region of hippocampus was not blocked by the Akt neutralizing antibody (Aktl/2 N-19). Whole-cell recordings of AMPAR-mediated EPSCs in C A 1 neurons of acute hippocampal slices were performed. To induce L T D in the C A 1 region of acute hippocampal slices, a pre-synaptic stimulation (1 Hz for 5 min) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -40 m V of C A 1 pyramidal neurons was used. A , Akt l /2 (N-19), when intracellularly applied, binds with Akt and blocks its function to phosphorylate its substrates. Normal IgG was used as control and normal intracellular solution without any drugs was used as blank control. B , representative traces of AMPAR-mediated EPSCs. 38 * Akt / x ? £ - 52 IB: phospho-(Ser/Thr) Akt substrate - 28 " 52 IB! GST mm---- — O O Figure 11. /« vitro Ak t phosphorylation o f G l u R l C T but not GluR2 CT. A , G S T - G l u R l CT, GST-GluR2 C T and G S T alone were incubated with recombinant active A k t l / P K B a (30 pg/ml) in normal Akt reaction buffer at 30 'C for 1 hour. B, In the experiment with 3 2 P detection, G S T - G l u R l CT, GST-GluR2 C T and G S T alone were incubated with recombinant active A k t l / P K B a (60 ug/ml) in the Akt reaction buffer with y - 3 2 P ATP. 39 Western blot experiments leaded us to identify Thr840 as a site for Ak t phosphorylation, and we also found that CaMKI I and P K C phosphorylation site Ser831 and P K A phosphorylation site Ser845 could not be phosphorylated by Akt (Fig. 12). Unexpectedly, no Thr840 phosphorylation was detected when Ser839 was mutated. A n explanation is that Ser839, which is so close to Thr840, may help to form a certain conformation which is required for Ak t phosphorylation on Thr840. Alternatively, this conformation may be important for recognition of Thr840 phosphorylation by the anti-phospho GluRl (T840) antibody. However, I could not exclude Ser839 as an Akt phosphorylation site, since, as shown in Fig. 12B, no phosphorylation was detected by the phospho-(Ser/Thr) Akt substrate antibody when either Thr840 or Ser839 was mutated. Furthermore, we also found that Thr840 is specific for Akt , and neither P K A (Fig. 13 A ) nor P K C (Fig. 13B, 13C) was able to phosphorylate it. Thus, our results identify a previously unrecognized phosphorylation site in the G l u R l C T which may be critical in facilitated membrane fusion-dependent A M P A R insertion during LTP. 4. G l u R l C T Thr840 phosphorylation during LTP in the hippocampal C A 1 region To examine whether the Thr840 phosphorylation on G l u R l C T is enhanced during LTP, hippocampal slice extracts were tested by western blot to measure the Thr840 phosphorylation levels with anti-phospho-GluRl(T840) antibody (1:400). LTP was induced by the same protocol as described in the experiments for F ig . 7. Slices were collected and homogenized in the homogenization buffer right after and 2, 5, 10, 20, 30 min after the application of LTP protocol, respectively. Slices without stimulation were 40 Akt o o IB: phospho-(Ser/Thr) Akt substrate K D — 49 — 35 — 28 B Akt j » . & c F ^ 6 s G O <^ o-N ^ IB:phospho-GluR1 (T840) Reprobe: phospho-(Ser/Thr) Akt substrate Reprobe: GST K D ~ 4 9 _ 35 — 49 "35 "49 "35 IB: phospho-GluR1 (S845) Reprobe: phospho-GluR1 (S831) Reprobe: G S T Akt A A r> C? C? O PKA o \ 6 & cT & c # <? KD -49 "35 - 4 9 35 - 4 9 35 Figure 12. /« vitro Akt phosphorylation of G l u R l C T on Thr840. G S T - G l u R l CT, GST-GluR2 C T and related mutated G S T - G l u R l C T (M831, M838, M839, M840 and M845) were incubated with recombinant active A k t l / P K B a (30 p.g/ml) in normal Akt reaction buffer at 30 'C for 1 hour. A , B, Akt phosphorylated G l u R l C T on Thr840. C , Akt phosphorylated neither Ser831 (CaMKI I and P K C phosphorylation site) nor Ser845 ( P K C phosphorylation site). 41 IB: phospho-GluR1 (S845) Reprobe: phospho-(Ser/Thr) Akt substrate Reprobe: phospho-GluR1 (S831) Reprobe: GST PKA Akt <*> <£6 <£° <# r / ( # # KD 49 35 -49 "35 ^ ^ ^ ^ ^ —49 —35 —49 — 35 B IB:phospho-GluR1 (S831; Reprobe: GST Akt PKC ^ ^ c / # V / ^ ^ KD - 49 - 3 5 ^ 9 — 3 5 IB: phosphor-(Ser/Thr) Akt substrate Reprobe: phospho-GluR1 (S831) Reprobe: phospho-GluR1 (S845) Reprobe: GST PKC Akt KD — 49 — 35 — 49 — 35 — 49 — 35 — 49 — 35 Figure 13. In vitro neither P K A nor P K C phosphorylated G l u R l C T on Thr840. G S T - G l u R l CT, GST-GluR2 C T and related mutated G S T - G l u R l C T (M831, M838, M839, M840 and M845) were incubated with recombinant active A k t l / P K B a (30 ug/ml) in normal Akt reaction buffer at 30 'C for 1 hour. A , P K A could not phosphorylate G l u R l C T on Thr840. B, C, P K C could not phosphorylate G l u R l C T on Thr840. 42 used as control. Another G l u R l C T antibody was used to detect the total G l u R l for normalization. A s shown in F ig . 14A, an increased phosphorylation of G l u R l on Thr840 occurred during the first 5 min after the LTP induction by H F S and lasted for less than 10 min. F ig . 14B shows that Thr840 phosphorylation level right after LTP stimuli was significantly increased compared to control, whereas no significant difference was shown 30 min after LTP stimuli. Thus, there was a fast and transient phosphorylation of G l u R l on Thr840 during LTP. To further confirm that this increased phosphorylation of G l u R l C T on Thr840 during LTP was solely caused by Akt , slices were incubated with several inhibitors separately for 15 min immediately prior to the H F S and then Thr840 phosphorylation levels were tested. I used PI3K inhibitor, wortmannin (10 uM), to block Akt activation since there is no specific Akt inhibitors commercially available. I also used P K A inhibitor, R p - c A M P S (25 uM), and P K C inhibitor, chelerythrine (10 uM), to test that whether P K A or P K C phosphorylates G l u R l C T on Thr840 during LTP in the hippocampal slices. In addition, N M D A R antagonist A P V (50 uM) was used to confirm that this enhanced Thr840 phosphorylation by Akt is NMDAR-dependent. Slices without H F S were used as negative control, while slices with H F S but without kinase inhibitor treatment were used as positive control. A s shown in F ig. 15, the enhancement of Thr840 phosphorylation on G l u R l C T during LTP was blocked or slightly blobked by A P V , wortmannin and chelerythrine but not by R p - c A M P s , indicating that Akt does phosphorylate G l u R l C T on Thr840 during LTP in the hippocampal slices and this process is NMDAR-dependent, whereas P K A cannot phosphorylate Thr840. It seems that P K C may have some effect on Thr840 phosporylation during LTP in the hippocampal 43 Time after LTP stimuli (min) in r» Control II 2 5 10 20 30 IB: Phospho-GluR1 (T840) IB: GluRl CT — 100 KD —100 KD Figure 14. G l u R l Thr840 phosphorylation in the C A 1 region of hippocampus during LTR To induce LTP, the high frequency stimulation (HFS, 3 trains of 100Hz) was applied to the Schaffer collateral fibers projecting to C A 1 pyramidal neurons. Slice extracts were tested by western blot to measure G l u R l Thr840 phosphorylation. A , A significant enhancement of G l u R l phosphorylation on Thr840 occurred during the first 5 min after the LTP stimuli and lasted for no more than 10 min. Slices without stimulation were used as control. B, normalized results showing mean phosphorylation of G l u R l Thr840 as mean±S.E. (n=8, A N O V A F=8.149, P O . 0 5 ) . *P<0.05 indicates significant difference from control. "Right after LTP stimuli" means that slices were collected and homogenized immediately after LTP stimuli. "30 min after LTP stimuli" means that slices were collected and homogenized 30 min after LTP stimuli. 44 Control' LTP APV PKAI PKCI PI3KI IB: phospho-GluR1 (T840) IB: GluRl CT — 100 KD — 100 KD B Control A P V PKAI PKCI PI3KI LTP Figure 15. Inhibition of G l u R l Thr840 phosphorylation in the C A 1 region of hippocampus during LTP by A P V and PI3K inhibitor. To induce LTP, the high frequency stimulation (HFS, 3 trains of 100Hz) was applied to the Schaffer collateral fibers projecting to C A 1 pyramidal neurons. Slice extracts were tested by western blot to measure G l u R l Thr840 phosphorylation. A , The enhancement of G l u R l phosphorylation on Thr840 was blocked by A P V and PI3KI but not by P K A I and P K C I . P K A I stands for P K A inhibitor, R p - c A M P S (25 uM); P K C I stands for P K C inhibitor, chelerythrine (10 uM). PI3KI stands for PI3K inhibitor, wortmannin (10 uM). The concentration of A P V was 50 u M . Slices without H F S were used as negative control, while slices with H F S but without kinase inhibitor treatment were used as positive control. B, normalized results showing mean phosphorylation of G l u R l Thr840 as mean±S.E. (n=8, A N O V A F=4.450, P<0.05). *P<0.05 indicates significant difference from control. 45 slices, and this needs further confirmation. 5. LTP in the hippocampal C A 1 region was not blocked by G l u R l C T peptides To answer the question whether the contribution of Akt to LTP results from its ability to phosphorylate G l u R l on Thr840, a synthesized peptide, G l u R l C T (832-844) peptide, was intracellularly used in our whole-cell recording experiment. If phosphorylation of G l u R l C T on Thr840 is required for LTP, this peptide, containing the amino acid sequence of 832-844 of G l u R l C T ( I N E A I R T S T g ^ L P R N ) (500 ug/ml), may be able to block LTP by competing with endogenous G l u R l as the substrate of Akt . The peptide with phosphorylation on Thr840 ( INEAIRTSpTg^LPRN) (500 ug/ml) and the scrambled peptide (500 ug/ml) were used as controls. The same LTP protocol, a pre-synaptic stimulation (2 H z for 100 s) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -5 m V of C A 1 pyramidal neurons, was used. A s shown in the F ig. 16, none of the peptides blocked LTP, indicating other mechanisms instead of phosphorylation of G l u R l on Thr840 may be responsible for A M P A R insertion and LTP. 46 Figure 16. LTP in the C A 1 region of hippocampus was not blocked by G l u R l C T (832-844) peptide. Whole-cell recordings of AMPAR-mediated E P S C s in C A 1 neurons of acute hippocampal slices were performed. To induce LTP in the C A 1 region of acute hippocampal slices, a pre-synaptic stimulation (2 H z for 100 s) to the Schaffer collateral fibers paired with a post-synaptic depolarization to -5 m V of C A 1 pyramidal neurons was used. A , G l u R l C T (832-844) peptide containing the amino acid sequence of 832-844 o f G l u R l C T (rNEArRTST 8 4 0LPRN) (500 ug/ml) was intracellularly applied. The peptide with phosphorylation on Thr840 (INEArRTSpT84oLPRN) (500 ug/ml) and the scrambled peptide (500 ug/ml) were used as controls. B, representative traces of AMPAR-mediated EPSCs . 47 D I S C U S S I O N 1. The role of Akt in regulating synaptic plasticity In our previous study, we have found that P I3K is required for A M P A R insertion during LTP (Man et al., 2003), so the present work was extended to its primary downstream protein kinase Akt. Akt is believed to be a very important protein kinase involved in many biological responses especially cell survival (Dudek et al., 1997; Datta et al., 1999), and its role in modulating synaptic plasticity is quite a recent area of research and not well clarified. A previous study in our lab found that direct phosphorylation by Akt is an important mechanism underlying G A B A A R translocation to the cell surface, which regulates the number of G A B A A R at post-synaptic sites (Wang et al., 2003). This provided the first evidence for the role of Ak t in controlling synaptic strength. In our present study, I found a significant increase of Akt activation during LTP, and with the application of Akt dominant negative peptide and Akt neutralizing antibody, I demonstrated that Akt is required for LTP in hippocampal C A 1 region. Furthermore, I identified Thr840 on G l u R l C-tail as a novel Akt phosphorylation site. This work further confirmed the contribution of Akt to synaptic plasticity, in which direct phosphorylation may be a common mechanism. In addition to these ligand-gated ion channels, Akt is also involved in the regulation of voltage-gated ion channels. It has been reported that Akt was implicated in the potentiation of N and L type voltage-gated calcium channels induced by insulin-like growth factor-1 (Blair and Marshall, 1997; Blair et al., 1999). A n involvement of Ak t in T G F Pi regulation of a calcium-activated potassium channel has 48 also been found (Lhuil l ier and Dryer, 2002). Although the detailed mechanisms in these two cases were not clear, all of the above studies together provided strong evidence suggesting Akt as a novel modulator for both ligand-gated and voltage-gated ion channels, and this modulation may result from a common phosphorylation mechanism. Thus, Akt may play an essential role in the regulation of neuronal excitability and synaptic plasticity which are intimately associated with both ligand-gated and voltage-gated ion channel functions. 2. The role of G l u R l phosphorylation in A M P A R insertion and LTP A s previously reported, many signaling pathways such as CaMKI I , P K A and P K C are implicated in LTP (Malenka et al. 1986; Mal inow at al., 1989; Lisman et al., 1997; L ing et al., 2002). Either C a M K I I or P K C phosphorylates G l u R l C T on Ser831 during LTP (Roche et al., 1996; Barria et al., 1997; Mammen et al., 1997). However, mutating Ser831 to an aspartate residue which mimics the phosphorylation was not sufficient to induce receptor insertion (Boehm and Mal inow, 2005). Furthermore, synaptic G l u R l insertion during LTP was not blocked when Ser831 was mutated to an alanine residue which prevents the phosphorylation on Ser831 (Hayashi et al., 2000; Boehm and Mal inow, 2005). P K A phosphorylates G l u R l C T on Ser845 (Roche et al., 1996), but this phosphorylation by P K A was also not sufficient to drive G l u R l into the synapse (Esteban et al., 2003). Recently, Mal inow group identified Ser818 as a P K C phosphorylation site on G l u R l and found that LTP-inducing stimuli gave rise to the phosphorylation on this site. However, mimicking the phosphorylation on Ser818 by mutating to an aspartate 49 residue had no effect on the synaptic G l u R l insertion (Boehm et al., 2006). Together, it seems that synaptic delivery o f G l u R l is a multiple regulated process in which several phosphorylation events are l ikely to occur and CaMKI I , P K A or P K C phosphorylation alone is not sufficient for its induction. So far, there is no explicit phosphorylation pattern in G l u R l which is sufficient for A M P A R insertion. It is very l ikely that there are other unknown phosphorylation sites in G l u R l which are critical for its synaptic insertion. From the database search results, we noticed that there is a candidate motif (NEArRTST84oLPRNSGA) for Akt phosphorylation on G l u R l C T and Thr840 is the most l ikely phosphorylation site. Indeed, in the present study, Thr840 was identified as a novel phosphorylation site which is solely phosphorylated by Akt but by neither P K A nor P K C . This provided another potential controlling site for receptor insertion. Although I have not established whether phosphorylation on Thr840 is responsible for synaptic G l u R l insertion, I speculate that this site may cooperate with other phosphorylation sites to control synaptic G l u R l incorporation. There are some concerns about this experiment. A s shown in F ig . 6, Thr838, Ser839 and Thr840 are three amino acids in series on the G l u R l CT . First concern is that the specificity of the antibodies I used may not be high enough to distinguish the phosphorylation of these three consecutive sites. Thus, other more specific methods of detection are needed for further confirmation. For example, the radioactive detection of Akt phosphorylation on G l u R l C T may be a good way to try since it has no specificity problems. Another concern is that, since the three sites are so close to each other, there is a possibility that mutation of any of the three sites w i l l affect the Ak t phosphorylation or the antibody recognition of the other two sites by possibly 50 changing the required conformation. This could be a reasonable explanation for the unexpected result in which no Akt phosphorylation on Thr840 was detected when Ser839 was mutated (Fig. 12B). According to the result, I cannot exclude the possibility that Ser839 may also be a phosphorylation site for Akt , and further examination is needed. 3. The mechanisms by which Akt contributes to LTP I have attempted to demonstrate that Akt plays an essential role in LTP due to its ability to phosphorylate G l u R l on Thr840. We introduced G l u R l C T (832-844) peptide as a dominant negative which may be able to compete with endogenous G l u R l as the substrate of Akt . However, as shown in F ig . 16, intracellular application o f this peptide with a concentration up to 500 u.g/ml failed to block LTP in the C A 1 region of hippocampal slices. This could be due to technical reasons by which the peptide might not diffuse into the neuron or not get to the target. More experiments with positive controls showing that this peptide does diffuse into the neuron and get to the target are needed. Another possible explanation is that a spatial conformation of G l u R l C T is required for the phosphorylation on Thr840 by Akt and this peptide may be too short to form such a recognizable conformation by Akt. Therefore, it was not able to competitively block endogenous G l u R l phosphorylation by Ak t and hence had no effect on LTP. Another possibility is that the contribution of Akt to LTP results from mechanisms other than G l u R l Thr840 phosphorylation. For example, Ak t may facilitate LTP through other downstream proteins. It has been recently reported that activation of the PI3K/Akt pathway may contribute to the mechanisms of synaptic plasticity and 51 memory consolidation by promoting cell survival v ia Forkhead transcription factor ( F K H R ) and protein synthesis via mammalian target of rapamycin (mTOR) in the dentate gyrus (Horwood et al., 2006). Activation of m T O R has been found to be PI3K-dependent in the hippocampal C A 1 region (Cammalleri et al., 2003), suggesting that activation of m T O R mediated by Ak t activation may occur in hippocampal C A 1 neurons as wel l . Furthermore, a considerable body of studies has demonstrated a role o f m T O R pathway in the late phase of LTP in the hippocampal C A 1 region (Tang et al., 2002; Cammalleri et al., 2003; Kelleher et al., 2004; Tsokas et al., 2005; Vickers et al., 2005). Thus, it is possible that Akt contributes to LTP via its downstream m T O R pathway. If so, it seems that Akt plays a role in LTP maintenance. Indeed, my result supported this possibility. A s shown in F ig . 9A, in the anti-Akt N-19 treated group, AMPAR-mediated E P S C s were enhanced within the first 10 min immediately after LTP stimuli, but after that this potentiation decreased to the basal level gradually. It seems that LTP could be induced but not be sustained. In addition, C R E B transcription factor can be phosphorylated by Akt on Ser l33, resulting in increasing transcriptional activation of C R E B and affinity of C R E B to its co-activator C R E (Du and Montminy, 1998). Since C R E B is thought to play an important role in LTP maintenance by triggering gene transcription and protein synthesis, this provides another possible mechanism by which Ak t contributes to LTP (Bourtchuladze et al., 1994; Impey et al., 1996; Schulz et al., 1999; Bozon et a l , 2003 Chen et al., 2003). However, Akt may also contribute to LTP induction or expression through other downstream pathways. A s we noticed, PI3K, the upstream protein of Akt , plays a controversial role in LTP. Different studies have shown that activation of P I3K is 52 involved in LTP induction (Opazo et al., 2003), expression (Sanna et al., 2002) and maintenance (Raymond et al., 2002) in the hippocampal C A 1 region. So it is very possible that Akt plays a multiple role in LTP since it has a wide influence on neuronal activity through many downstream pathways. 4. Future direction Some studies have revealed a common mechanism by which Akt exerts its biological actions by directly phosphorylating its substrate proteins and creating a phosphorylation-dependent protein-protein interaction motif (Datta et al. 2000; Tziv ion and Avruch, 2002). The identified Akt phosphorylation site around Thr840 (RTSTg4oLP) on the G l u R l C T has a high degree of homology to 14-3-3 binding motif " R S X ( p S / T ) X P or R X X X ( p S / T ) X P " (where R is arginine, S is serine, T is threonine, P is proline and X is any amino acid) (Tzivion and Avruch, 2002; Fujita et al., 2002). 14-3-3 is a small (-30 kD) adaptor protein that forms both homo- and heterodimers (Bridges and Moorhead, 2005) and has been considered as an active cofactor in cellular regulation by S/T phosphorylation (Datta et al., 2000; Tziv ion and Avruch, 2002), which is implicated in diverse cellular functions including stimulation of secretory exocytosis (Morgan and Burgoyne, 1992; Roth et al., 1993). It has been reported that 14-3-3 readily binds phosphorylated Ser413 on the active-zone protein R T M l a , contributing to LTP of granule cell-Purkinje cell synapses in the mouse cerebellum (Simsek-Duran et al., 2004). This study provided the first evidence for the involvement of 14-3-3 in regulating synaptic plasticity. There is a possibility that 14-3-3 may bind to G l u R l following Akt 53 phosphorylation on Thr840 and trigger the following actions. Indeed, our preliminary experiment supported this possibility. This pointed out a direction for further study which may provide a breakthrough in our understanding of how A M P A R insertion is facilitated during LTP. 5. Significance of the present study The physiological implications of the present work is not only limited to modulation of AMPAR-mediate synaptic transmission. Since P B K / A k t is a well-known signaling pathway mediating cell survival (Dudek et al., 1997; Datta et al., 1999), Akt could be the pivot connecting LTP and cell survival. M y findings support the possibility that induction of the LTP form of synaptic plasticity may have significant neuroprotective action through P B K / A k t pathway. 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