Author's Accepted Manuscript

Molecular Regulation of Synaptogenesis

during Associative Learning and Memory
Thomas J. Nelson, Daniel L. Alkon

www.elsevier.com/locate/brainres

PII: S0006-8993(14)01660-6
DOI: http://dx.doi.org/10.1016/j.brainres.2014.11.054
Reference: BRES43958

To appear in: Brain Research

Accepted date:
25 November 2014

Cite this article as: Thomas J. Nelson, Daniel L. Alkon, Molecular

Regulation of Synaptogenesis during Associative Learning and Memory,
Brain Research, http://dx.doi.org/10.1016/j.brainres.2014.11.054

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Molecular Regulation of Synaptogenesis during
Associative Learning and Memory
Thomas J. Nelson and Daniel L. Alkon

Blanchette Rockefeller Neurosciences Institute

Morgantown, WV 26505 USA

Corresponding author:
Thomas J. Nelson
BRNI
8 Medical Center Drive
Morgantown, WV 26505 USA
Phone 1-304-293-0930
Email tjnelson@brni-jhu.org

Keywords: Learning; Memory; Synaptogenesis; Protein Kinase C; Synapse; BDNF

Highlights:

• Biochemical pathways in adult synaptogenesis are described.

• Synaptogenesis plays a central role in associative learning and memory.

• Cell-cell contact and soluble factors released from astrocytes are important in synaptogenesis.

• Intracellular signaling pathways involving protein kinase C provide neuronal specificity which is

required for associative memory.



ABSTRACT

Synaptogenesis plays a central role in associative learning and memory. The biochemical pathways that

underlie synaptogenesis are complex and incompletely understood. Nevertheless, research has so far

identified three conceptually distinct routes to synaptogenesis: cell-cell contact mediated by adhesion

proteins, cell-cell biochemical signaling from astrocytes and other cells, and neuronal signaling through

classical ion channels and cell surface receptors. The cell adhesion pathways provide the physical

substrate to the new synaptic connection, while cell-cell signaling may provide a global or regional signal,

and the activity-dependent pathways provide the neuronal specificity that is required for the new synapses

to produce functional neuronal networks capable of storing associative memories. These three aspects of

synaptogenesis require activation of a variety of interacting biochemical pathways that converge on the

actin cytoskeleton and strengthen the synapse in an information-dependent manner.

1. Introduction

Recent research has shown that synaptogenesis is not only important during development, but also plays a

central role in associative learning and memory. Synapse loss is found in patients with major clinical

depression and in neurodegenerative disorders such as Alzheimer's disease, where it is associated with

decreased memory and cognitive function. Conversely, antidepressants produce significant increases in

the number of synapses in the adult brain. Synaptogenesis can be triggered by neuron-astrocyte or neuron-

neuron contact, and mediated by cell-adhesion proteins including neurexin/neuroligin, Eph receptors, and

cadherins, which activate intracellular signaling pathways involving cofilin, GTPases, and other

pathways. It can also be triggered by soluble factors released from astrocytes, including BDNF,

thrombospondins, and apolipoprotein E/cholesterol. For synaptogenesis to have a meaningful connection

to associative learning and memory, a third process of synapse stabilization and maturation, which

depends on neuron-specific synaptic activity, is also important. This process involves long-term changes

to neuronal connectivity and cytoarchitectural changes mediated by intracellular proteins including



protein kinase C, Rho GTPases, and cofilin, which convert unstable filopodia to stable mushroom

synapses that can survive for the lifetime of an individual. Drugs that enhance synaptogenesis, such as

protein kinase C activators or TrkB receptor agonists, may be useful in restoring normal neuronal function

in a variety of disorders.

2. Cell adhesion proteins

Synapses are structural elements between cells, so synaptogenesis necessarily involves the formation of

new cell-cell contacts (Fig. 1). These trans-synaptic contacts are mediated by extracellular interactions

between a variety of adhesion proteins including neuroligins, neurexin, cadherins, ephrin, SynCAMs, and

leucine-rich-repeat proteins.

2.1. Neuroligins

Neuroligins are postsynaptic cell adhesion proteins that form complexes with presynaptic neurexin

dimers. Neuroligin-deficient mice have normal density of synaptic contacts, but their synapses show

decreased spontaneous GABAergic and glutamatergic activity indicative of dysfunctional synapses

(Varoqueaux et al., 2006). Neuroligin overexpression increases the number of synapses (Dean and

Dresbach, 2006) and RNA interference knockdown of neuroligin-1, -2, and -3 reduces the number of both

excitatory and inhibitory synapses (Chih et al., 2005).

Presynaptically, neurexins interact with CASK (calcium-calmodulin-dependent serine protein kinase),

Band4.1 actin binding protein (Biederer and Sudhof, 2001), and Mint/X11, which are involved in

formation of multiprotein complexes. These protein complexes trigger reorganization of actin

cytoskeleton.

2.2. Cadherins

N-cadherins (Togashi et al., 2002) are calcium-dependent adhesion proteins that are essential for normal

dendritic spine density (Saglietti et al., 2007). Cell-cell adhesive interactions mediated by a variety of cell



adhesion molecules are early events in synaptogenesis, and cell-surface localization of cadherins is

essential in the stabilization of new synapses. Membrane-associated guanylate kinase proteins, such as

DLG5 (Disc Large-5), stabilize cadherin-catenin complexes at the plasma membrane (Nechiporuk et al.,

2007). DLG5 interaction with ȕ-catenin regulates synaptogenesis (Wang et al., 2014). N-Cadherin is a

focal point of several signaling pathways including Rho GTPases and phosphoinositide 3-kinase

(Stepniak et al., 2009; Arrikath and Reichardt, 2008). The scaffold protein afadin (Beaudoin et al., 2012)

binds cadherins and is also essential in synaptogenesis.

2.3. PSD-95

Another member of the DLG family is PSD-95, a postsynaptic density scaffolding protein that interacts

with NMDA receptors and numerous signaling proteins to regulate dendritic spine and synapse density

(Kim and Sheng, 2004; Feng and Zhang, 2009, Migaud et al., 1998; El-Husseini et al., 2000; Béïque et

al., 2006; Vickers et al., 2006). The function of PSD-95 is to accelerate synaptic maturation by enhancing

postsynaptic clustering of AMPA glutamate receptors (El Husseini et al., 2000), and by stabilizing early

synaptic contacts (Taft and Turrigiano, 2013). It associates with cell adhesion molecules such as

neuroligins (Irie et al., 1997) and netrin-G1 ligand (NGL-1) (Lin et al., 2003; Ricciardi et al., 2012). PSD-

95 is frequently used as a postsynaptic marker of synapses.

Many other trans-synaptic adhesion proteins interacting with PSD-95 have been described. The PSD95-

interacting postsynaptic adhesion protein netrin-G ligand-3 interacts with leukocyte common antigen-

related (LAR) protein, which is a receptor tyrosine phosphatase. This interaction has been shown to

regulate the formation of excitatory synapses in cultured rat hippocampal neurons (Woo et al., 2009).

2.4. Leucine-rich repeat proteins and SynCAMs

Leucine-rich repeat proteins constitute another class of postsynaptic synapse-organizing transmembrane

proteins (de Wit and Ghosh, 2014). An example is LRRTM2 (leucine rich repeat transmembrane neuronal

protein 2), a protein expressed on the surface of neuronal cells, which binds to neurexins on the



presynaptic side of neurons, producing cell adhesion junctions. In this respect LRRTM2 functions like the

postsynaptic protein neuroligin. As for neuroligin, binding of LRRTM2 to neurexin induces presynaptic

differentiation. When artificially expressed in neurons or non-neuronal cells in culture, LRRTM2

competes with neuroligin-1 for neurexin-1ȕ and potently induces synapse formation onto these cells (Ko

et al., 2009). This forms the basis of the 'artificial synapse formation' assay, which is useful for screening

other synaptogenic proteins (Linhoff et al., 2009).

Another important cell adhesion protein is the immunoglobulin family protein SynCAM (Biederer et al.,

2002). SynCAM 1 and 2 form homo- and heterophilic adhesive interactions, attaching pre- and post-

synaptic membranes together at the earliest stages of synapse formation. SynCAM increases the number

of excitatory synapses, and is required for maintaining normal synapse numbers. These synapses are

functional and highly plastic; overexpression of SynCAM 1 in adult mouse brain improves spatial

learning in the Morris water maze task (Robbins et al., 2010).

2.5. Eph receptors

NMDA receptors and Eph receptors regulate synaptogenesis both during development and in the mature

brain. Cofilin, which is a member of the actin-depolymerizing-factor (ADF/cofilin) family (described

below), promotes spine retraction (Zhou et al., 2012), which may preserve the capacity for spine

remodeling in the mature brain (Murai et al., 2003). There are two forms of Eph, and they have

complementary effects: EphA activates cofilin, while EphB inhibits it. EphB functions by modulating the

guanine nucleotide exchange factors (GEFs) intersectin and T-cell lymphoma invasion and metastasis-

inducing protein 1 (Tiam1) and Kalirin, which activate the small GTPases Cdc42 and Rac, respectively

(Tolias et al., 2007). Activation of the EphB receptor modifies calcium influx by inducing

phosphorylation of the NMDA receptor (Dalva et al., 2000).

Ephrin-EphB interactions also participate in postsynaptic-to-presynaptic reverse signaling (Lim et al.,

2008; Li et al., 2011) at two points: first by increasing neurotransmitter release, and second by enhancing



postsynaptic glutamate responsiveness (Hruska and Dalva, 2012). NMDA receptors are relatively more

important in immature synapses. As synapse maturation progresses to the mature so-called mushroom

synapse, Į-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPA) receptor signaling

becomes more important (Wu et al., 1996; Hall and Ghosh, 2008). Calcium-calmodulin-dependent

protein phosphorylation is required to consolidate mature spine morphology (Matsuzaki et al., 2004). This

effect is dependent on actin cytoskeletal rearrangement (Matsuzaki et al., 2004) (Fig. 2).

3. Synaptogenic factors secreted from astrocytes

In cultured neuronal and retinal cells, astrocyte-conditioned medium contains a variety of substances

secreted from astrocytes that induce synaptogenesis. One of these was identified as a lipoprotein complex

of apolipoprotein E (apoE) and cholesterol (Mauch et al., 2001; Ullian et al., 2001; Ullian et al., 2004;

Fester et al., 2009; Goritz et al., 2005). The primary source of cholesterol is cholesterol synthesized in

neurons and astroglia. Little or none crosses the blood-brain barrier (Pardridge and Mietus, 1980). Thus,

under conditions when cholesterol availability is rate-limiting, cholesterol from glia, which is imported

through the low-density lipoprotein receptor, would increase the rate of growth.

3.1. Apolipoprotein E

Several mechanisms have been suggested for cholesterol stimulation of synaptogenesis. Fester et al.

(2009) found evidence that conversion to estradiol may be required. Estradiol also stimulates the release

of glial apoE (Struble et al., 2007). Goritz et al. (2005) suggested that cholesterol directly induces

synaptic differentiation. Other reports (Elmariah et al., 2005) indicate that adding free cholesterol has no

effect on synaptogenesis, suggesting that integration into ApoE-containing LDL particles is important.

ApoE3, but not apoE4, was found to protect cultured hippocampal neurons against ȕ-amyloid oligomers

(Sen et al., 2012), consistent with suggestions that the E3 form is more effective at neuronal repair and

neuroprotection. ApoE3 can induce expression of protein kinase Cİ mRNA (Sen et al., 2012), which is a

key signaling protein involved in memory (Alkon et al., 1988).



Although the primary function of apoE is as a cholesterol-transporting molecule, in the brain it also

functions as a repair molecule. Intraneuronal apoE is greatly increased after brain injury (Horsburgh et al.,

2000). ApoE is synthesized primarily by astrocytes under normal conditions (Mahey et al., 2006), but

can also be released by neurons under conditions of injury or stress (Huang and Mucke, 2012; Mahey et

al., 2006). Following neuronal injury and deafferentation, HMG-CoA reductase activity is depressed in

both astrocytes and neurons, while apoE is synthesized and released Poirier et al., 1991). Conversely,

during dendritic remodeling and synaptogenesis, neuronal synthesis of apoE is suppressed and

internalization of cholesterol through the apoE/LDL receptor pathway is favored (Poirier, 1996) Lesions

of the entorhinal cortex produce compensatory synaptogenesis that is abolished in apoE knockout mice

(Masliah et al., 1995).

3.2. BDNF

The neurotrophin BDNF plays a central role in synaptogenesis (Fig. 2), dendritic growth (McAllister et

al., 1995) and neurorepair (Lu et al., 2013). BDNF levels in the brain are regulated at the DNA, RNA, and

protein levels. BDNF increases the spine density of hippocampal CA1 pyramidal neurons in postnatal

hippocampal slices (Tyler and Pozzo-Miller, 2003), increases spine motility (Horch et al., 1999), induces

new thin spines, and enlarges the size of mushroom spines (Ji et al., 2010).

BDNF mediates synaptic plasticity induced by environmental enrichment (Novkovic et al, 2014), fear

conditioning (Heldt et al., 2014, Mizuno et al, 2012), long-term potentiation (Yamada and Nabeshima,

2003), one-trial associative learning (Bekinschtein et al, 2007), classical eyeblink conditioning (Keifer et

al, 2009) and spatial maze learning (Mizuno et al., 2003). Classical aversive conditioning increases

expression of mature BDNF in pigeon hippocampus (Faria et al., 2013). However, the details of its

mechanism of action are still unclear. BDNF was reported to reverse the impairment of memory produced

by the protein synthesis inhibitor anisomycin, suggesting that one requirement of protein synthesis, long

known to be essential in associative memory, may be to produce additional mature BDNF (Moquel-



González, 2008). In rats, contextual fear conditioning, another form of associative learning, triggered high

levels of BDNF expression in a subpopulation of glutamatergic neurons in hippocampal CA1 region

(Chen et al., 2007). This synthesis is regulated by neuronal activity (Greenberg et al., 2009). The reverse

is also true: BDNF regulates transcription at the dendrite (Santos et al., 2010).

BDNF is sufficient to rescue L-LTP when protein synthesis is inhibited (Pang et al, 2004). By injecting

antibodies to BDNF and antisense oligonucleotides to the CA1 region, it was found that BDNF synthesis

was required after training for persistence, but not formation, of inhibitory avoidance training memory

(Beckinschtein et al, 2007), consistent with the idea that BDNF and protein synthesis are primarily

involved in downstream long-term consolidation events. BDNF activates transcription of a immediate-

early genes including c-fos and Zif268 (Beckinschtein et al, 2007).

The Bdnf gene contains eight promoters and multiple splicing and polyadenylation sites that result in 18

different transcripts, all of which produce an identical BDNF protein (Aid et al., 2007). Most of these

promoters are regulated by neuronal activity (Hong et al., 2008). At the mRNA level, BDNF mRNA

stability is increased by phosphorylation of coactivator-associated arginine methyltransferase 1

(CARM1), an enzyme that methylates proteins involved in mRNA stability, including the RNA-binding

protein HuD (ELAV, embryonic lethal abnormal vision protein 4). Phosphorylation of CARM1 by PKC

inactivates it, eliminating its ability to methylate and thereby inactivate HuD. PKC phosphorylation of

the resulting demethylated HuD enhances its binding to neurotrophin precursor messenger RNAs, and

stimulates dendritic maturation of cultured hippocampal neurons (Lim and Alkon, 2012) (Fig. 3).

BDNF is also induced by physical exercise, environmental enrichment (Ambrogini et al., 2013), and

traumatic brain injury (Grundy et al., 2000), suggesting a role in neuronal repair. After translation BDNF

undergoes multiple steps of proteolytic cleavage, from preproBDNF to proBDNF and finally to mature,

fully processed BDNF. Although mature BDNF is known to play a critical role in long-term potentiation

and memory, the role of proBDNF has been controversial. ProBDNF may be important in long-term



depression (Pang et al., 2004), and there is evidence that proBDNF promotes synapse elimination, while

mature BDNF inhibits it (Je et al., 2013). There is also evidence that proBDNF binds to sortilin and

induces neuronal apoptosis (Teng et al., 2005).

A number of proteolytic enzymes can convert proBDNF to mature BDNF, including furin, PACE4, and

protein convertase PC5/6B (Seidah et al, 1996). However, tissue plasminogen activator (tPA)-dependent

activation of plasminogen (Pang et al, 2004) was essential for late-phase long-term potentiation in mouse

hippocampus, suggesting that tPA is the physiological catalyst.

Alternative polyadenylation sites result in long 3'UTR BDNF transcripts localized to dendrites and short

3'UTR transcripts localized to cell soma (An et al., 2008). These different sourced BDNFs have different

functions. Long 3'-UTR BDNF is translated locally in the dendrites; knockdown of dendritic long 3'-UTR

Bdnf mRNA blocks spine head enlargement and spine elimination, while knockdown of somatic short 3'

UTR Bdnf mRNA blocks spine formation (Orefice et al., 2013). The BDNF synthesized in dendrites

remains as proBDNF; thus these differences support the idea that proBDNF is a synapse elimination

factor and that processing to mature BDNF occurs in the cell soma. BDNF synthesis has also been

reported in microglial cells (Yang et al, 2012) and astrocytes (Lu et al, 2014).

The downstream actions of BDNF and TrkB receptors are mediated by the PI3K/Akt pathways,

ERK/Ras, and phospholipase CȖ pathways (Ikegaya et al., 2002; Xia et al., 2010; Leal et al., 2014). The

protein kinase Akt /PKB is involved in several apoptosis-inhibiting pathways. Akt, BDNF, and the

number of dendritic spines in mouse hippocampus are all increased by environmental enrichment,

resulting in cognitive enhancement and neurogenesis (Ramirez-Rodruguez et al., 2014).

3.3. BDNF and Protein kinase C

The phospholipase CȖ (PLCȖ) pathway of BDNF is particularly significant because the products of PLCȖ

are inositol 1,4,5-trisphosphate (IP3), which is a major calcium-releasing signal, and 1,2-diacylglycerol,

which is an activator of protein kinase C (PKC) signaling. PKC is involved in synaptic activity-induced



signaling (Bank et al., 1988; Olds et al., 1989), and this synaptic activity provides the neural network

specificity that is required for associative memory. By amplifying or attenuating the signaling pathway in

different neurons, PKC performs essential gating and focusing functions for synaptogenic signals such as

BDNF and apoE. As mentioned above, PKCİ stabilizes HuD, which increases the stability and rate of

translocation of target mRNAs. HuD increases as a result of PKC activation after learning (Pascale et al.,

2004) and stabilizes the mRNA for BDNF, nerve growth factor (NGF), and neurotrophin-3 (NT-3) (Lim

et al., 2012). Thus, BDNF induces regenerative-type amplification of its own synaptogenic activity in a

manner dependent on neuronal signaling.

BDNF increases the levels of KIF1A (Kondo and Hirokawa, 2012), a kinesin-related protein involved in

transport of N-methyl-D-aspartate receptor subunit 2B (NMDAR-2B) (Yin et al., 2011). Knockdown of

Kif1a prevented environmental enrichment from inducing hippocampal synaptogenesis (Kondo and

Hirokawa, 2012) and memory impairment (Yin et al., 2011). Although BDNF is produced in both

astrocytes and neurons, evidence indicates that BDNF from neurons has a direct effect on synaptogenesis,

while astrocytes modulate synapse formation indirectly by stimulating Trk signaling between neurons

(Elmirah et al., 2005). BDNF is also required for activity-dependent maintenance of mature spine, while

blocking BDNF reduces spine density, increases spine length and decreases spine head width (Kellner et

al., 2014).

3.4. Thrombospondins

Another set of astrocyte-derived factors, thrombospondins 1 and 2 (Christopherson et al., 2005), is also

important in synaptogenesis. Application of thrombospondin 1 to cultured retinal ganglion cells or

embryonic neurons produces a large increase in the number of excitatory synapses (Xu et al., 2010). The

new synapses are postsynaptically silent (Christopherson et al., 2005; Xu et al., 2010), and require other

as-yet unidentified factors to become functional. Thrombospondin 1 increases the rate of synaptogenesis,

but has no effect on the final number of synapses (Xu et al., 2010). Thrombospondin binds to neuroligins,



which are postsynaptic synaptic adhesion proteins (see above). The Alzheimer's disease ȕ-amyloid protein

inhibits thrombospondin release from astrocytes (Rama Rao et al., 2013).

Hevin (SC1) is another synaptogenic glycoprotein produced by astrocytes. Like thrombospondin, hevin

also produces postsynaptically silent excitatory synapses in cultured rat retinal ganglion cells

(Kucukdereli et al., 2011). Hevin is related to the Secreted Protein Acidic and Rich in Cysteine (SPARC)

protein, which acts antagonistically to hevin and blocks synapse formation.

Glypicans 4 and 6 (Allen et al., 2012) are proteins released from astrocytes that induce functional

synapses between retinal ganglion cells (Allen et al., 2012). These molecules increase the surface

clustering of Į-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and glutamate receptors.

Glypican 4 is enriched in the hippocampus and glypican 6 is enriched in the cerebellum. Tumor necrosis

factor-Į (TNF-Į) also increases GluR1 AMPA receptor quantal amplitude, nonspecifically increasing

general synaptic efficacy, in a process called synaptic scaling (Steinmetz and Turrigiano, 2010).

An important question is how synaptogenesis and synaptic maturation are related. The extracellular

matrix contains numerous substances that stabilize neuronal connectivity and decrease plasticity (de Vivo

et al., 2013). Enzymatic digestion of chondroitin sulfate proteoglycans, which are abundant in the

extracellular matrix, increases synaptic plasticity by decreasing the amount of physical restriction on

dendritic spines (Pizzorusso et al., 2002), suggesting that non-protein components exert a significant

restraint on whether memories are protected or destabilized. Thus, treatment of hippocampal slices with

matrix metalloproteinase-9, an extracellular protease required for LTP maintenance, was found to activate

the ȕ-integrin pathway, which inactivated cofilin, leading to actin polymerization (Wang et al., 2008).

4. Neuronal signaling pathways

Transduction of adhesion protein signals activates several neuronal signaling pathways that ultimately

result in stabilization of the synapse to its mature mushroom morphology. These structural changes

involve signaling pathways such as Rho GTPases and protein kinase C that modify the cytoskeleton.



4.1. Rho GTPases

Rho GTPases mediate signaling between cell surface receptors and N-cadherins, which regulate

cytoskeletal morphology (Tolias et al., 2011). In excitatory glutamatergic synapses the receptors are

NMDARs and mGluRs. Rho GTPases include RhoA, Rac1, and Cdc42 (Ridley, 2006). Rac1 and Cdc42

are involved in spine formation and growth, while RhoA is involved in spine loss (Newey et al., 2005).

Activity of Rho GTPases determined by the relative activity of guanine nucleotide exchange factors

(GEFs), which enhance GDP-GTP exchange, and GTPase-activating proteins (GAPs), which accelerate

GTP hydrolysis (Rossman et al., 2005). Specificity is determined by specific GEFs and GAPs. For

example, Rac GAPs include Į1-, Į2-, ȕ1-, and ȕ2-chimaerin (Sosa et al., 2009). Į1-Chimaerin is found in

dendritic spines (Buttery et al., 2006). Į1-Chimaerin is rapidly ubiquitinated and degraded under basal

conditions, but is stabilized by C1 domain binding molecules such as 1,2-diacylglycerols or phorbol

esters (Marland et al., 2011). In the presence of phorbol ester, Į1-chimaerin is recruited to the membrane

and binds to the N2A subunit of the NMDA receptor, where it inactivates Rac, inhibiting its dendrite-

pruning activity (Van den Ven et al., 2005).

Rac GEFs such as Kalirin 7 are essential for the activity of EphA receptors, which are large tyrosine

kinases that control dendritic spine maturation (Penzes & Jones, 2008). Other Rac GEFs, such as Tiam1,

Kalirin, and Trio (Buttery et al., 2006), are localized in dendritic spines and post-synaptic densities. Tiam

1 binds to the NMDA receptor, which promotes dendritic arborization (Tolias et al., 2005).

Rho GTPases affect synaptic function by activating p21-activated kinases (PAKs) and Rho-kinases

(ROCKs). PAKs and ROCKs phosphorylate LIM-kinases (LIMKs) and slingshot phosphatases (SSHs),

which modify the phosphorylation state of cofilin.

4.2. Cofilin

Cofilin-1 is a protein in the actin-depolymerizing factor (ADF) family that directly controls actin

assembly and disassembly (Bernstein and Bamburg, 2010). When cofilin becomes phosphorylated on



Ser-3 by Src, TES (testicular protein kinases), or LIMKs (Mizuno, 2013), it is inactivated and ceases to

bind G-actin (monomeric, globular actin) and F-actin (filamentous actin) (Bernstein and Bamburg, 2010).

Inactive cofilin is sequestered by the 14-3-3 protein, a PKC substrate long suspected of being involved in

memory. Ultimately, dephosphorylated cofilin becomes ubiquitinated and is degraded by the proteasome.

It is not, however, totally inert, and can activate phospholipase D1, which hydrolyzes phosphatidylcholine

to choline and phosphatidic acid, a PKC activating lipid. In cardiac tissues, phospholipase D1 is involved

in adenosine anti-ischemic signaling pathways involving binding to RhoA GTPase (Mozzicato et al.,

2003), but in neurons phosphatidic acid produced by PKD1 is reported to negatively modulate dendritic

branching (Zhu et al., 2012).

Dephosphorylation of cofilin-1 by phosphatases SSH or chronophin (Gohla et al., 2005) activates binding

of cofilin to mature actin. This binding can be inhibited by inorganic phosphate. Active cofilin is a potent

nucleator of actin polymerization, but the effects of cofilin on actin are very complex: it may promote

actin depolymerization of polymerization depending on the cofilin-actin ratio and on the presence or

absence of other regulatory proteins. At low cofilin-actin ratios, cofilin produces persistent severing of F-

actin. At high ratios, it converts F-actin into a twisted form by transiently filament severing. Twisted actin

is unstable in the absence of cofilin, and stabilizes in a more stable untwisted form a few hours later. At

very high, nonphysiological ratios, or when cells are stressed, cofilin forms actin-cofilin bundles in which

the cofilin is unable to function and thus becomes functionally inactive (Bernstein and Bamburg, 2010).

By catalyzing the disassembly of actin, cofilin increases the concentration of G-actin and barbed ends and

thereby stimulates accelerates actin filament polymerization, paradoxically resulting in increased actin

turnover (Hotulainen et al., 2005, Kiuchi et al., 2007).

Cofilin competes with the Arp2/3 complex, which creates branch points in the formation of dendritic

structures characteristic of lamellipodia (Robinson et al., 2001). Cofilin also de-branches actin, undoing

the branching activity of Arp2/3. In many cells Arp2/3 and cofilin act sequentially to produce cyclic

branching and debranching.



Cofilin is also inhibited by binding phosphatidylinositol 4,5-bisphosphate (Yonezawa et al., 1990) and

cortactin (Oser et al., 2009). It is activated by actin-interacting protein (Ono, 2003) and cyclase-associated

protein (Moriyama and Yahara, 2002). Slingshot phosphatase (SSH 1L), which dephosphorylates and

activates cofilin, is itself activated by calcineurin (a calcium-dependent protein phosphatase) and inhibited

by phosphorylation by calmodulin-dependent protein kinase (CaMKII), an abundant PSD protein (Zhao et

al., 2012). CaMKII forms a complex with SSH1L and 14-3-3 (Zhao et al., 2012).

4.3. Protein Kinase C

Direct astrocyte-pyramidal neuron contact through integrins causes the release of arachidonic acid from

PL2A and activation of PKC, which in turn promotes maturation of excitatory postsynaptic terminals

(Hama et al., 2004). PKC also mediates the effect of ApoE signaling in cultured hippocampal neurons

(Sen et al., 2012), suggesting that PKC may have a dual effect. In fact, activation of PKCİ and Į by

bryostatin or activation of PKCİ by the isozyme-specific activator DCPLA methyl ester produces

synaptogenesis in cell culture and in mice (Hongpaisan et al., 2011, 2013), indicating that PKC activation

alone can be sufficient to trigger synaptogenesis.

Bryostatin 1 is a PKCİ and Į activator that binds to the C1A and C1B domains on PKC (DeChristopher et

al., 2012). The physiological ligand for the C1 domain is 1,2-diacylglycerol (Blumberg et al., 2008).

Some PKC isoforms, including conventional (Į, ȕI, ȕII, and Ȗ), and novel PKC isoforms (į, İ, Ș, and ș)

also possess a C2 domain, which binds anionic lipids such as phosphatidic acid and phosphatidylserine

(Corbalán-Garcia et al., 2003; Conesa-Zamora et al., 2001). These lipids are part of the cell membrane

and come into contact with PKC after it is translocated from the cytosol by binding to a C1 ligand such as

1,2-diacylglycerol or bryostatin. Pharmacological activation of PKC with bryostatin or DCP-LA methyl

ester enhances memory and associative learning by increasing the numbers of mushroom spines,

perforated postsynaptic densities, and double-synapse presynaptic boutons associated with spines

(Hongpaisan et al., 2011, 2013). These effects are mediated primarily by the İ isoforms of PKC (Nelson



et al., 2009). Specific inhibitors of PKC, such as Ro 31-8220, prevent these effects, supporting the

hypothesis that activation of PKC may be an important trigger for synaptogenesis (Hongpaisan and

Alkon, 2007). This is consistent with previous findings that PKC becomes activated and translocated in

mammalian hippocampus after rabbit nictitating membrane associative learning (Bank et al., 1988) and

rat water maze training (Olds et al., 1989). PKC activation also markedly enhances associative learning in

invertebrates such as Hermissenda crassicornis (Etcheberrigaray et al., 1992; Alkon et al., 1982; Kuzirian

et al., 2006), indicating that this pathway is well conserved during evolution.

Activation of PKC by C1 ligands is transient, lasting only 15-30 minutes before returning to baseline.

However, the signaling and transcriptional changes can last for days. Application of bryostatin to cultured

mammalian cells produced an increase in protein 35S labeling that lasted for >3 days (Alkon et al., 2005).

Bryostatin also induced the synthesis of other proteins when given several days before associative training

in Hermissenda (Alkon et al., 2005). This decreased the number of training events required for memory

acquisition and extended retention from 7 minutes to over one week, suggesting that systemic PKC

activation can indeed act to enhance memory acquisition and consolidation.

Natural C2 ligands including phosphatidylserine and polyunsaturated fatty acids such as arachidonic acid

are involved in neurite outgrowth and spine formation (Ammar et al., 2013; Shirai et al., 2010). Artificial

C2 ligands, such as DCPLA methyl ester, can restore mature mushroom spine synapses and prevent

memory loss in aging rats (Hongpaisan et al., 2011). Despite a shorter t½ of dissociation, these ligands

produce a more prolonged effect and, unlike C1 activators, C2 activators also do not produce measurable

PKC downregulation, suggesting that PKC may have two different modes of activation with different

time courses of relaxation.

4.4. PKC, Actin and Cofilin

Appreciation for the role of degradative and destabilizing processes in memory represents a change from

the earlier picture of simple growth. Nowhere is this more true than in our understanding of the



complexity of actin dynamics at the synaptic spine. Cytoskeletal reorganization, involving destabilization

of actin, is an integral feature of synapse formation and memory (Hotulainen and Hoogenraad, 2010).

Dendritic spines are highly dynamic; over 80% of the F-actin in spines is reported to turn over every

minute (Calabrese and Halpain, 2005). Actin associates with numerous proteins, including MARCKS

(Myristoylated Alanine-Rich C-Kinase Substrate), an acidic membrane-bound protein. PKC

phosphorylates MARCKS, inhibiting the association between MARCKS and actin and with the plasma

membrane. This reduces the ability of MARCKS to crosslink F-actin, and triggers dendritic spine

destabilization (Calabrese and Halpain, 2005). Thus, interactions between actin and signaling kinases

such as PKC can modify the geometry of dendritic spines, changing the interconnectivity of neuronal

networks responsible for cognition.

Depolymerization of F-actin causes synapse loss (Zhang and Benson, 2001). Actin recruits other proteins,

such as Piccolo, to the active zone of synapses (Chia et al., 2014). Cell adhesion molecules are critical

components of synapse formation. SYG-1, for example, binds to the WAVE regulatory complex (WRC),

leading to loss of local F-actin and axonal branches (Chia et al., 2014). Guidance proteins such as GAP-

43/B-50/neuromodulin and netrin are also important in synapse formation. GAP-43 is a growth cone-

associated protein that promotes the stabilization of long actin filaments (He et al., 1997).

5. Similarities and Differences between Associative Learning and Long-Term Potentiation

Many of the same biochemical pathways in learning are also important in long-term potentiation (LTP),

and studies of LTP and long-term depression (LTD) have greatly improved our understanding of synaptic

plasticity. Both require CaM-dependent protein kinases and PKC (Lisman et al., 2002), Eph receptors

(Klein, 2009; Trabalza et al., 2014), and protein synthesis, and are strongly regulated by BDNF

(Minichiello, 2009). Both involve reorganization of the actin cytoskeleton in dendritic spines (Jedlicka et

al., 2008; Baudry et al., 2012) that is mediated by ADF/cofilin phosphorylation and dephosphorylation

(Gu et al., 2010; Meng et al., 2003). Both involve RNA-binding proteins (Pfeiffer and Huber, 2006). Both



activate CA3-CA1 synapses (Gruart et al., 2006), and most compounds that disrupt synaptic integrity

interfere both in LTP and associative learning.

One-trial inhibitory avoidance learning produces changes rat hippocampus similar to those observed in

LTP, including phosphorylation of the GluR1 subunit of the L-alpha-amino-3-hydroxy-5-

methylisoxazole-4-propionate (AMPA) receptor and enhancement of field EPSPs in the CA1 region,

suggesting that learning induced LTP (Whitlock et al., 2006). In general, however, LTP has not yet been

correlated with learning in live animals. While learning may sometimes induce LTP, it has been difficult

to demonstrate whether LTP can induce learning. Recently it was reported that animals could be

conditioned to associate foot shock with a high-frequency optogenetic depolarizing stimulus that induces

LTP (Nabavi et al., 2014). Low frequency stimulation inactivated the memory and high frequency

stimulation reactivated it, indicating that stimuli that induce LTP and LTD can produce behaviorally

relevant conditioned responses. While these findings are exciting, they have not resolved the debate about

whether LTP is an essential part of learning or whether LTP is both necessary and sufficient for learning.

The earlier findings showing that spatial learning can occur, at least in some circumstances, without

NMDA receptor-dependent LTP (Saucier and Cain, 1995; Bannerman et al., 1995), still pose a challenge.

Biochemically, there are clear similarities but also differences between LTP and associative learning,

especially in ion channels. Gene targeting of the GluR-A subunit of the AMPA receptor abolishes LTP

without affecting spatial learning (Zamanillo et al., 1999). By contrast, deletion of GluR-B impairs spatial

memory but not LTP (Shimshek et al., 2006). Antisense knockdown of the Kv1.4 potassium channel

Mice eliminates early- and late-phase LTP and reduced paired-pulse facilitation (which are presynaptic

effects) while having no effect on spatial memory (Meiri et al., 1998). Mice deficient in NELL2, a

thrombospondin-like extracellular protein, exhibit spatial learning impairment (Matsuyama et al., 2005)

but enhanced LTP (Matsuyama, 2004). MPC17742, a selective NMDA antagonist, and 1S,3S-ACPD, a

group II metabotropic glutamate receptor agonist, block LTP but not spatial learning (Maru, 2001). Mice

lacking metabotropic glutamate receptor 5 (mGluR5) have normal CA3 LTP but impaired CA1 LTP and



spatial learning (Lu et al., 1997). Since the mossy fiber pathway in CA3 LTP is independent of NMDA

receptors, it suggests that NMDA signaling may play a more important role in LTP than in associative

memory. Nonetheless, it is clear that LTP and memory share many important features (Fig. 4).

6. Conclusion

Adult synaptogenesis and dendritic spine formation and maturation may be the principal physiological

substrates for associative memory. The biochemical pathways that underlie these processes are complex

and incompletely understood. Nevertheless, research has so far identified three pillars of synaptogenesis:

cell-cell contact mediated by adhesion proteins, cell-cell biochemical signaling from astrocytes and other

cells, and neuronal signaling through classical ion channels and cell surface receptors, mediated by

calcium and signaling molecules such as PKC. However, this tripartite distinction is only conceptual,

because the components of synaptogenesis all influence each other in influencing actin dynamics.

During development, large numbers of excess synapses are created. Creation of these synapses depends

on spontaneous patterned activity in the developing brain, which produces waves of depolarization and

calcium transients, and may use many of the same molecular pathways as described here (Cline, 2001).

Unnecessary and redundant synaptic connections are later eliminated postnatally in a process that requires

retrograde signaling by semaphorins (Uesaka et al., 2014). Understanding these processes may provide

valuable clues about how synapses are lost in neurodegenerative disorders.

Actin dynamics are equally important in adults for retraction of unused dendritic spines and focusing of

dendritic branching, which are essential aspects of synaptic plasticity. Synaptic pruning is also a critical

step during brain development, and pruning deficits caused by loss of macroautophagy have been

implicated in autistic disorders (Tang et al., 2014). Nevertheless, widespread synapse loss in adults is a

pathological phenomenon, and is recognized as one of the earliest pathological features in Alzheimer's

disease (Masliah et al., 1990; Lassmann et al., 1993) and possibly other dementias (Clare et al., 2010).



Thus, the pathways that regulate synaptic morphology and function are certain to become primary targets

for pharmacological intervention and remain so for the foreseeable future.

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Figure Legends

Fig. 1. Pathways involved in synaptic contact formation.

Fig. 2. Trans-synaptic pathways for synaptogenesis. Neurexin-neuroligin binding triggers presynaptic

differentiation and postsynaptic actin reorganization through actin-binding protein CASK. EphrinB-EphB
signaling increases the motility of immature filopodia and forms adhesive interactions in mature dendritic
spines. Other cell adhesion proteins, including cadherin, synCAMs, and leucine-rich-repeat proteins (not
shown) also stabilize the new synapse. EphB can also be on the presynaptic side where its interaction with
postsynaptic EphrinB can trigger presynaptic differentiation in so-called reverse signaling. Soluble factors
from astrocytes, including apolipoprotein E/cholesterol complex, BDNF, and thrombospondins act on
PKC, neuroligins, and intracellular signaling pathways to induce spine maturation.

Fig. 3. PKC activation of neurotrophin translation. PKC phosphorylates and inactivates Coactivator-
Associated Arginine Methyltransferase 1 (CARM1), preventing it from methylating HuD protein. PKC



also increases the affinity of HuD for neurotrophin mRNAs, stabilizing them and increasing their level of
expression (Lim and Alkon, 2012).

Fig. 4. Similarities and differences between LTP and associative learning.



Highlights:

• Biochemical pathways in adult synaptogenesis are described.

• Synaptogenesis plays a central role in associative learning and memory.

• Cell-cell contact and soluble factors released from astrocytes are important in synaptogenesis.

• Intracellular signaling pathways involving protein kinase C provide neuronal specificity which is

required for associative memory.



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