Mol Neurobiol DOI 10.1007/s12035-014-8768-8

The Role of Phosphoinositides in Synapse Function Yoshibumi Ueda

Received: 14 December 2013 / Accepted: 1 June 2014 # Springer Science+Business Media New York 2014

Abstract Since the discovery of phosphatidylinositol-3-kinase, scientific interest in the biological functions of phosphoinositides has greatly increased. Currently, seven phosphoinositides have been identified. These phosphoinositides are specifically localized to organelle membranes, their site of action. Phosphoinositides can regulate neuronal function by specifically recruiting downstream proteins that have phosphoinositidebinding domains. To date, it is well accepted that phosphoinositides play important roles in a broad spectrum of neuronal functions from regulating neural development to modulating synapse function. This review will provide an overview of the function and distribution of phosphoinositides at synapses. Keywords Phosphoinositide . Phosphatidylinositol 3,4,5-trisphosphate . Spinule . Synaptic plasticity . Förster resonance energy transfer . Fluorescence lifetime imaging Abbreviations PIP3 Phosphatidylinositol 3,4,5-trisphosphate PI(4,5)P2 Phosphatidylinositol 4,5-bisphosphate PI(3,4)P2 Phosphatidylinositol 3,4-bisphosphate PI(3,5)P2 Phosphatidylinositol 3,5-bisphosphate PI(4)P Phosphatidylinositol 4-phosphate PI(3)P Phosphatidylinositol 3-phosphate PI(5)P Phosphatidylinositol 5-phosphate PI3K Phosphatidylinositol 3-kinase PI4K Phosphatidylinositol 4-kinase PI(4)P5K Phosphatidylinositol 4-phosphate 5-kinase Y. Ueda (*) Department of Hematology and Immunology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Kahoku, Ishikawa 920-0293, Japan e-mail: [email protected] Y. Ueda e-mail: [email protected]

PI(5)P4K PI FRET AMPAR NMDAR CaMKII LTP LTD SHIP IP3 ER FAPP2 PTEN Fig4 MTMR OCRL INPP4A PLIP OSBP CERT

Phosphatidylinositol 5-phosphate 4-kinase Phosphatidylinositol Förster resonance energy transfer α-Amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor N-methyl-D-aspartate receptor Ca2+/calmodulin-dependent protein kinase II Long-term potentiation Long-term depression Src homology 2-containing inositol 5′phosphatase Inositol 1,4,5-trisphosphate Endoplasmic reticulum PI(4)P adaptor protein 2 Phosphatase and tensin homolog Factor-induced gene Myotubularin-related protein 2 Oculocerebrorenal syndrome of Lowe Inositol polyphosphate 4-phosphatase 4a PTEN-like phosphatase Oxysterol-binding protein Ceremide transfer protein

Introduction The brain is comprised of billions of neurons that form intricately interconnected functional networks, which mediate higher order functions such as learning and emotional processing. Neurons are highly polarized cells with distinct compartments, including the cell body, axon, dendrites, and synapses, specialized junctions where inter-neuronal communication occurs. Neurons also have the unique ability to adapt their activity to different external inputs, a phenomenon known as plasticity. The cooperative interplay between

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different signaling molecules and proteins is key for neuronal plasticity. How are complicated signals computed by neurons? One hypothesis is that compartmentalized signaling by cellular membranes is critically involved [1]. Neurons (as well as other cells) contain many membranous compartments such as the plasma membrane, mitochondria, endoplasmic reticulum (ER), Golgi apparatus and endosomes, which are all important sources of lipid membrane. Membranes serve as a place where signaling proteins can interact and initiate subsequent downstream signaling. Signaling proteins are recruited with high temporal precision to membranes by lipid second messengers. Phosphoinositides are one important group of lipid second messengers, characterized by an inositol ring and a glycerol backbone with two fatty acids (Fig. 1a). The diverse phosphorylation of D-3, D-4, and D-5 positions on the inositol ring gives rise to seven phosphoinositides species. Each phosphoinositide has a distinct localization within neurons and interacts with different types of proteins to enable compartmentalized signal transduction.

Emergence of Phosphoinositides Phospholipid signaling was revealed by the discovery of phospholipid turnover following acetylcholine stimulation in pigeon pancreas and guinea pig brain by Hokin and Hokin [23]. Subsequent studies revealed that phospholipid signaling is a universal signaling mechanism that can be evoked by a diverse range of growth factors and neurotransmitters. Furthermore, intense research lead to the identification of phosphatidylinositol 4,5bisphosphate (PI(4,5)P2) as the source of phospholipid turnover [24]. PI(4,5)P2 can be cleaved by phospholipase C (PLC) into two further products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) [24]. IP3 increases Ca2+ through IP3 receptors on the ER. The increase in cytosolic Ca2+ leads to further Ca2+ release through ryanodine receptors on the ER. The Ca2+ increase induces Ca2+ entry through Orai channels at the plasma membrane [25]. IP3 signaling is involved in the regulation of a wide range of cell functions such as neural development, cell proliferation, fertilization, and sensory perception [26]. Studies from the Nishizuka group revealed that DAG works as an activator of protein kinase C (PKC) and is implicated in cellular functions such as cell differentiation and proliferation [27–29]. Thus, phosphoinositide turnover is important for a broad spectrum of signaling pathways and essential for the maintenance of cell function. A series of studies by the Martin and Holz groups in the early 1990s suggested that PI(4,5)P2 itself is required for secretion in chromaffin cells [30–32], which provided great contribution in the area of membrane trafficking. Additionally, in 1994, it was revealed that PI(4,5)P2 binds directly to the pleckstrin homology (PH) domain that a broad spectrum of signaling proteins occupy [33]. This evidence leads to the idea

that PI(4,5)P2 is not only the precursor of DAG and IP3, but can also function as a lipid second messenger. In 1988, the Cantley group reported that type I phosphatidylinositol kinase (PIK) specifically phosphorylates the D-3 ring position of the inositol moiety to generate a novel phospholipid, known as phosphatidylinositol 3-phosphate (PI(3)P) [6]. This report spearheaded the discovery of a new family of phosphoinositides, all of which are phosphorylated at the D-3 position on the inositol ring [6]. This included the isolation of phosphatidylinositol 3,4,5-trisphosphate (PIP3) in formyl–methionyl–leucyl–phenylalanine (fMLP)-activated neutrophils by the Sklar group (also in 1988) [34] and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2), again by the Cantley group in 1989 [35]. Additionally, in 1997, the Cantley group identified phosphatidylinositol 5-phosphate (PI(5)P), a phosphoinositide with phosphate on the D-5 position [36]. In the same year, the Ulug group discovered phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), a phosphoinositide with phosphate residues on both D-3 and D-5 positions [37]. Hence, the decade from 1988 to 1997 saw the discovery of many phosphoinositides and is viewed as a golden era for the field of lipid research.

Characteristic Properties and Species of Phosphoinositides Currently, seven phosphoinositides have been identified. The discovery of these different phosphoinositides variants highlights the diverse characteristics of phosphoinositides. Investigations (in mostly non-neuronal cells) have revealed insights into the relative abundance of different phosphoinositides (Fig. 1b; for an extensive review, see ref. [2]). The most abundant phosphoinositides are phosphatidylinositol 4phosphate (PI(4)P) and PI(4,5)P2 [2]. While the amount of PI(3,4)P2, PI(3,5)P2, PI(5)P and PIP3 are maintained at relatively low levels under basal conditions, stimulation can cause a transient increase in the amount, indicating that phosphoinositide levels are tightly regulated by neuronal activity [5, 38–40]. However, recent studies have revealed that an aberrant constitutive increase of phosphoinositides can induce neuronal dysfunction. For example, an abnormal elevation in PI(4,5)P2 levels at nerve terminals can increase the ratio of clathrin-coated vesicles in all synapses and reduce synaptic transduction [41]. The accumulation of PI(3,4)P2 induces excitotoxic neuronal death [17]. Additionally, phosphatase and tensin homolog (PTEN)-deficient mice, where PIP3/Akt signaling is constantly active can lead to Cowden syndrome, a condition characterized by hypertrophic and ectopic dendrites and axonal tracts, enhanced synaptogenesis, and behavioral changes including abnormal social interaction [42]. PI(3,5)P2 accumulation, on the other hand, causes Charcot–Marie–Tooth disease, a

Mol Neurobiol Fig. 1 The characteristics of phosphoinositides. a The general structure of phosphoinositides. b The relative amount of phosphoinositides. PI(4)P, PI(4,5)P2, PI(3,5)P2, and PI(3,4)P2 values are referred from ref. [2]. PI(3)P and PI(5)P values are calculated from refs. [3, 4]. The value for PIP3 is taken from ref. [5]. c PI is converted to PI(3)P by PI3K classes II and III (also known as Vps34) [6, 7], to PI(5)P by PIKfyve [8, 9], and to PI(4)P by PI4K [10]. PI is generated from PI(3)P by MTMR2 [11]. PI(3)P is converted to PI(3,5)P2 by PIKfyve [9]. PI(4)P is converted to PI(4,5)P2 by PI(4)P5K [10]. PI(5)P is converted to PI(4,5)P2 by PI(5)P4K [8, 13]. A PTENrelated 5-phosphatidylinositol phosphatase (PLIP) is a potential producer of PI from PI(5)P [14, 15]. PI(4,5)P2 is converted to PIP3 by PI3K [16] and PI(4)P by synaptojanin and OCRL [12]. PI(3,4)P2 is degraded to PI(3)P by INPP4a [17]. PIP3 is degraded to PI(4,5)P2 by PTEN [18] and to PI(3,4)P2 by SHIP [19], OCRL and group IV 5-ptase [20]. PI(3,5)P2 is converted to PI(5)P by MTMR2 [11, 21]. PI(3,5)P2 is degraded to PI(3)P by Fig4 [22]. PI is predominantly localized at endoplasmic reticulum, colored in yellow. PI(4)P is localized at Golgi apparatus in non-neuronal cells and synaptic vesicle in neurons colored in orange. PI(5)P, PI(3,5)P2, and PI(3)P function at the endosome highlighted in pink, while PI(3,4)P2, PI(4,5)P2, and PIP3 mainly work at plasma membrane colored in green

a

b

O

O O

O

HO P HO

6

1

2

3

OH OH 5

4

OH

PI PI(4)P PI(4,5)P2 PI(3)P PI(5)P PI(3,4)P2 PI(3,5)P3 PIP3

1000 50 50 2.5 0.5-2.5 0.4-0.8 0.4-0.8 0.05

c PI(5)P

MTMR2

PI(3,5)P2

Endoplasmic reticulum Golgi apparatus

PIKfyve

PLIP

Fig4

PIKfyve

Plasma membrane

PI3K classII, III

PI

Endosomal compartments

PI(3)P MTMR2

PI(5)P4K

PI4K

INPP4a

PI(4)P Synaptojanin OCRL

PI(3,4)P2 SHIP OCRL GroupIV 5-ptase

PI(4)P5K PI3K classI

PI(4,5)P2

PIP3 PTEN

DAG

severe hereditary motor and sensory neuropathy characterized by focally folded myelin sheaths and demyelination [21, 22]. These studies indicate that the cellular levels of phosphoinositides must be tightly regulated, as excessive amounts are strongly associated with disease. Therefore, understanding the enzymes involved in the synthesis and degradation of phosphoinositides is the key to homeostatically controlling the levels. The synthesis pathway and cellular localization of phosphoinositides were also investigated mainly using non-

IP3

neuronal cultured cells. The main precursor of these phosphoinositides is phosphatidylinositol (PI). PI is primarily synthesized from CDP-DAG and inositol by phosphatidylinositol synthase in the endoplasmic reticulum (ER) [43], and then transported to other organelles via lipid transfer proteins or vesicular trafficking (as shown in glucosylceramide recently [44]). PI is subjected to phosphorylation at each organelle where the specific enzymes responsible for phosphorylation at 3, 4, and/or 5 positions on the inositol ring are located. Several enzymes have been identified for each phosphoinositide

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(Fig. 1c). The localization and function of different phosphoinositides is outlined in the following sections. PI(4)P PI(4)P is generated from PI by phosphatidylinositol 4-kinase (PI4K) [45–51] and is predominantly localized to the Golgi apparatus in non-neuronal cells [52, 53]. PI(4)P is important for a wide range of cell functions including trafficking from the Golgi apparatus to the endosome and non-vesicular lipid trafficking. Golgi apparatus to endosome trafficking is regulated by the binding of PI(4)P to clathrin adaptors such as epsinR [54] and AP-1 [55]. Additionally, PI(4)P works as an anchor for lipid-transfer proteins such as ceramide transfer protein (CERT) [56], oxysterol-binding protein (OSBP) [57], and PI(4)P adaptor protein 2 (FAPP2) [44]. For instance, CERT binds to the PH domain of PI(4)P, which promotes localization to the Golgi apparatus. The START domain in CERT can pull ceramide out of the ER and transport it to the Golgi apparatus presumably at ER-Golgi contact site [56]. Likewise, FAPP2 and OSBP regulates the transport of glucosylceramide from Golgi apparatus to the plasma membrane and cholesterol from the ER to Golgi apparatus, respectively. In neuronal cells, the function of PI(4)P on Golgi apparatus in the postsynaptic compartment remains unclear. However, at the presynaptic side, a report demonstrates that PI4K activity is concentrated at synaptic vesicles [45]. Additionally, synaptojanin, a protein involved in the dephosphorylation of PI(4,5)P2 to PI(4)P, is also present at the presynaptic side and plays an important role in the endocytosis of synaptic vesicles [53, 58]. These studies suggest that PI(4)P is localized at synaptic vesicles. PI(4)P is also produced by dephosphorylation from PI(4,5)P2 by oculocerebrorenal syndrome of Lowe (OCRL), which is responsible for the oculocerebrorenal syndrome. The dysregulation of PI(4,5)P2 metabolism to PI(4)P leads to several neurological diseases. OCRL mutation causes severe cognitive defects [59]. As for synaptojanin, PI(4,5)P2 metabolism is altered in Down syndrome model mice that have trisomic synj 1. Disomic treatment of synj 1 mice alleviated the aberrant PI(4,5)P2 metabolism and improved cognitive disabilities, suggesting that synaptojanin 1 may contribute to brain dysfunction and cognitive disabilities in Down syndrome [60]. PI(4,5)P2 PI(4)P is converted to PI(4,5)P2 by phosphatidylinositol 4phosphate 5-kinase (PI(4)P5K) [10]. PI(4)P5K isozymes consist of PI(4)P5Kα, β, and γ [10]. PI(4)P5Kγ is enriched in neurons [61]. PI(4,5)P2 production is also performed by PTEN from PIP3 [18] and by phosphatidylinositol 5-phosphate 4kinase (PI(5)P4K) from PI(5)P [8, 13]. Considering that PIP3 and PI(5)P are less abundant than PI(4,5)P2, PI(4)P could be

the main source of PI(4,5)P2. However a new function for PI(4)P has recently been identified, which differs from its role as a precursor for PI(4,5)P2 [62]. Using a specific probe, a study showed that PI(4,5)P2 was enriched at the plasma membrane of the soma, axons, dendrites, and spines of cultured hippocampal neurons [63]. However, it is considered that PI(4,5)P2 levels locally increase more [64]. The PIP2 can regulate endocytosis, exocytosis, and actin polymerization (described in later parts) by recruiting appropriate proteins through PH, ENTH, C2 domain, and so on as shown in Table 1. PIP3 PIP3 regulates a broad spectrum of neuronal functions, such as dendritic arborization [115], cell size [119], and axonal filopodia formation [120], as well as the function of synapses [121]. PIP3 is rarely present under basal conditions. However, a small amount of PIP3 is required for α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor (AMPAR) clustering in spines [122]. PIP3 is generated from PI(4,5)P2 by class I phosphatidylinositol 3-kinase (PI3K) [16]. While PI3K is expressed throughout the neuron [123], the active form of PI3K (which is formed following the association with AMPAR is mainly localized to spines [124]. PIP3 is dephosphorylated by Src homology 2-containing inositol 5′-phosphatase (SHIP), OCRL, and group IV 5-ptase to PI(3,4)P2, and by PTEN to PI(4,5)P2. One report indicates that PTEN is mainly localized to the dendritic shaft [125]. The distinct localization of PTEN and active PI3K could determine where PIP3 accumulates in spines. Consistent with this idea, using a fluorescence lifetime-based PIP3 probe, our lab has shown that PIP3 is highly enriched in spines (compared to the dendritic shaft) of hippocampal CA1 pyramidal neurons [126]. Recently, PIP3 is also found at the presynaptic side and regulates syntaxin1A clustering and neurotransmitter release [123]. PI(5)P PI(5)P was the last member of the phosphoinositide family to be discovered and very little is known about its regulation and function. In non-neuronal cells, subcellular fractionation experiments revealed that PI(5)P is predominantly present in the plasma membrane [4]. PI(5)P is also observed in the smooth endoplasmic reticulum and Golgi apparatus to some extent [4]. PI(5)P can be generated by several different pathways. PIKfyve, the main enzyme involved in catalyzing PI(3)P to PI(3,5)P2, can also produce PI(5)P from PI [3, 8]. Another report shows that myotubularin-related protein 2 (MTMR2) can generate PI(5)P from PI(3,5)P2 [11, 21, 127, 128]. PI(5)P degradation is conducted by PI(5)P4K, an enzyme that converts PI(5)P to PI(4,5)P2. There are three isoforms of PI(5)P4K: PI(5)P4Kα, β, and γ [129]. PI(5)P4Kα is localized at the cytosol, while PI(5)P4Kβ is enriched in nucleus. The

Mol Neurobiol Table 1 A comprehensive list of neuronal phosphoinositide-binding proteins Name

Target

Binding domain

Function

Reference

EEA1 PIKfyve SNX13 Profilin

PI(3)P PI(3)P PI(3)P PI(4,5)P2

Excitatory synaptic transmission Degradation of Ca(V) 1.2 channels, endocytic cycling of AMPAR Neural development Spine morphology

[65] [66–68] [69] [70]

Cofilin N-WASP Gelsolin Talin Ezrin Radixin

PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2

FYVE FYVE PX Positive electrostatic potential 40 % surface of cofilin PH N and C terminus FERM FERM FERM

AMPAR trafficking Development of dendritic spines and synapses Ca2+ channel and NMDAR activities Molecular interactions underlying synaptic junctions Filopodial protrusion formation, growth cone guidance Filopodial protrusion formation, growth cone guidance

[71] [72] [73] [74] [75–77] [76, 77]

Moesin Filamin α-Actinin MARCKS Cortactin Spectrin PLCγ Synaptotagmin Doc2 Munc13 Rabphilin 3A CAPS Mint Piccolo/ aczonin RIM Syntaxin1

PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2

FERM Amino acid basic region Amino acid basic region Amino acid basic region Amino acid basic region PH PH C2 C2 C1 C2 C2 PTB C2

Filopodial protrusion formation, growth cone guidance Migration of neurons Spine morphology, NMDAR activity Maintenance of dendritic spine, LTP Dendritic spine formation, growth cone Spine morphogenesis, axonal transport of mitochondria Neuronal migration Ca2+ sensor for the exocytosis Ca2+ sensor for asynchronous exocytosis Priming factor for synaptic vesicle Priming factor for synaptic vesicle Priming factor for synaptic vesicle Priming factor for synaptic vesicle Priming factor for synaptic vesicle

[76, 77] [78] [79, 80] [81, 82] [83, 84] [85, 86] [87] [88] [89] [90] [91] [92] [93] [94]

PI(4,5)P2 PI(4,5)P2

Priming factor for synaptic vesicle SNARE protein for synapse fusion

[95, 96] [97]

Epsin AP180 CALM AP-2 (α,β2,μ2) Arf 6

PI(4,5)P2 PI(4,5)P2 PI(4,5)P2 PI(4,5)P2

Creating membrane curvature Clathrin-mediated Endocytosis Clathrin-mediated Endocytosis Clathrin-mediated Endocytosis

[98] [99] [100] [101]

PI(4,5)P2

Clathrin-mediated Endocytosis

[99]

Dynamin

PI(4,5)P2

C2 Electrostatics and hydrophobic A/ENTH A/ENTH A/ENTH Electrostatics and hydrophobic Electrostatics and hydrophobic PH

[102–105]

PLD Preso KCNQ

PI(4,5)P2 PI(4,5)P2 PI(4,5)P2

Scission of vesicles at presynaptic side, and AMPAR cycling at endocytic zone in spines Neurite outgrowth Spine morphology and dendritic outgrowth Regulate cell membrane potential and excitability in neurons

Formin ARNO SHC Tiam1

PI(3,5)P2 PIP3 PIP3 PIP3

Polarization of neurons Regulation of dendritic development Neuronal migration, Regulation of hippocampal synaptic plasticity Dendritic spine development

[110] [111] [87, 112] [113]

Sos Akt/PKB Protrudin

PIP3 PIP3, PI(3,4)P2 PIP3, PI(3,4)P2, PI(4,5)P2

LTP induction Dendritic arborization Neurite outgrowth

[114] [115] [116, 117]

PH FERM Electrostatics and hydrophobic PTEN domain PH PTB PH PH PH FYVE

[106] [107, 108] [109]

For a comprehensive review on the proteins that function in other cell types (as well as additional information about neuronal cells) please refer to refs. [53, 118]

Mol Neurobiol

localization of PI(5)P4Kγ is unclear. The enzymatic activity of PI(5)P4Kα is much higher than PI(5)P4Kβ [129]. Additionally, PTEN-related 5-phosphatidylinositol phosphatase (PLIP) is a potential negative regulator of PI(5)P, causing degradation to PI [15]. Currently, several PI(5)P-specific binding domains have been identified. Dok-1, Dok-2, Dok-4, and Dok-5 selectively bind to PI(5)P through tandem domains of phosphotyrosine-binding (PTB) and pleckstrin homology (PH) domains [130, 131]. ING1, ING2, and ACF are nucleus-localized proteins, which are also associated with PI(5)P via the plant homeodomain (PHD) [8]. A report also indicates that ING2 regulates p53 acetylation via PI(5)P association [132]. In neurons, MTMR2 is enriched in spines. Knockdown of MTMR2 leads to a decrease in EEA1 clusters in dendrites. Hence, PI(5)P is presumably present at early endosomes [133]. In another report, PI(5)P4K was mostly present in vesicular compartments of the somatic cytoplasm of hippocampal neurons [13]. PI(3,4)P2 PI(3,4)P2 is generated from PIP3 upon activation of SHIP, OCRL, and group IV 5-ptase [19, 20] and dephosphorylated by inositol polyphosphate 4-phosphatases 4a (INPP4a) to PI(3)P [17]. INPP4a is deficient in Weeble mutant mice that exhibit significant neuronal loss in the cerebellum and hippocampal CA1 field, indicating that abnormal PI(3,4)P2 accumulation could cause neuronal dysfunctions [134]. Additionally, another study shows that PI(3,4)P2 regulates the number of Nmethyl-D-aspartate receptors (NMDARs) at spines, indicating that PI(3,4)P2 is enriched around NMDAR in spines [17]. There are two isoforms of SHIP: SHIP1 and SHIP2. The importance in SHIP is extensively studied in immunology. For example, SHIP1 KO mice leads to a severe myeloproliferative disorder and impaired NK cell function [135]. A study using migrating MDCK cells demonstrated that PI(3,4)P2 levels is higher in the front than in the rear of the cell, presumably mediating SHIP activation [136]. PI(3,4)P2 binds to downstream-signaling proteins including Akt and PDK1 via the PH domain, and TAPP1 and TAPP2 via the tandem PH domain containing protein (TAPP) domain. Currently, there are few reports about SHIP in neurons. However, SHIP2 downregulates PIP3 signaling by degrading PIP3 to PI(3,4)P2 upon nerve growth factor-induced neuritogenesis in PC12 cells [19]. PI(3)P PI(3)P regulates a broad range of cellular functions such as endomembrane membrane fusion [137], vesicular trafficking [138], and autophagy [139]. PI(3)P is produced from several pathways. PI3K class II produces PI(3)P at the plasma membrane and clathrin-coated vesicles in response to hormones [140]. PI3K class III, also known as Vps34, produces PI(3)P

at endomembranes [141]. Additionally, INPP4a dephosphorylates PI(3,4)P2, generating PI(3)P [17]. Furthermore, PI(3)P is produced by FIG4 from PI(3,5)P2 [22]. PI(3)P accomplishes its functions by recruiting downstream signaling proteins through different binding modules including the FYVE and PX domains. For instance, early endosome antigen-1 (EEA1) is localized at early endosomes through a FYVE domain and also recruits Rab5, leading to the fusion of early and late endosomes [137]. In hippocampal neurons, PI(3)P is primarily localized to dendrites and axons and partially to spines [142]. PI(3,5)P2 PI(3,5)P2, in combination with PI(3)P, regulates multiple aspects of endo-membrane trafficking in neuronal cells [142]. PI(3,5)P2 is generated from PI(3)P by PIKfyve and degraded to PI(3)P by FIG4 [9]. Interestingly, it is known that FIG4 and PIKfyve interact indirectly via an associated regulator of PIKfyve (ArPIKfyve) [143]. The complex is attached on the endosome through a EYVE domain and functions to regulate PI(3,5)P2 levels. In non-neuronal cells, PI(3,5)P2 is widely known for its role in regulating membrane trafficking, vacuole formation, and the function of endosomes and lysosomes [143, 144]. In neurons, PIKfyve regulates NMDA-induced voltagegated Ca2+ channel internalization [66] and postsynaptic trafficking of AMPAR to the plasma membrane [67].

Phosphoinositide-Binding Proteins in Neurons In the past, PI(4,5)P2 was considered just as a precursor for DAG and IP3. However, it was revealed that PI(4,5)P2 can bind to the pleckstrin homology (PH) domain of other proteins [33]. Since then, several new functions for PI(4,5)P2 have emerged. Studies have indicated that PI(4,5)P2 can directly interact with other proteins via the PH domain, to recruit specific signaling proteins to the membrane and enable control of downstream signaling pathways. Currently, it is well known that a variety of signaling proteins contain a phosphoinositidebinding domain. Table 1 shows a list of the signaling proteins reported to function in neurons. However, further intense examination is still required to determine how the interaction between phosphoinositides and the binding domains regulates neuronal function. For comprehensive reviews on phosphoinositide-binding proteins, please see refs. [53, 118].

Techniques for the Detection of Cellular Phosphoinositides Early studies utilised biochemical methods to measure cellular levels of phosphoinositides. Typical experiments involved the metabolic labeling of phosphoinositides with radioisotopes

Mol Neurobiol

(e.g., 14C, 3H, and 32P), followed by tissue homogenization and then extraction. In some cases, the lipids were subjected to the deacetylation of phosphatidylglycerol structures or the methylation of phosphates on the inositol ring after extraction for better separation of structurally similar lipids using highpressure lipid chromatography (HPLC; even without radioisotopes [145]). Recent advances in liquid chromatography and mass spectrometry analysis have greatly contributed to our ability to analyze lipids [5, 146–148]. These methods allow us to measure the absolute values of phospholipids and to obtain the lipid profile depending on the difference in fatty acid composition. When combined with fractionation techniques, this method enables the measurement of phosphoinositide levels at specific organelle membranes [148]. One report using synaptosome fractions showed that PI(4,5)P2 levels can be measured as detergent soluble and insoluble membranes [149]. However, the main drawback of these methods is that one cannot obtain the time course or single-cell profile of phosphoinositide dynamics. In addition, when subcellular distribution of phosphoinositides (Fig. 2) is

Fig. 2 Subspine distribution of phosphoinositides. PI(3)P is enriched in spines [142]. PI(3,5)P2 and PI(5)P is localized at endosomal compartments [133]. Additionally PI(3,5)P2 is involved in GluR1 synaptic vesicle trafficking [67]. PI(4)P is localized at synaptic vesicles because PI4K activity is detected [45]. PI(4,5)P2 is localized at the plasma membrane of the soma, axons, dendrites, and spines [63]. PI(3,4)P2 regulates the number of NMDARs at the synapse, indicating that PI(3,4)P2 is enriched around NMDAR in spines [17]. PIP3 regulates AMPAR clustering [122] and is found at presynaptic side, regulating SNARE [123]. Additionally, synaptotagmin is able to associate with PIP3 at basal condition [150]

examined by fractionation, the contamination from one membrane fraction to another should be taken into consideration. In order to observe phosphoinositides in single cells, fluorescent imaging tools were developed. Since the late 1990s, genetically encoded fluorescent protein-linked lipid-binding domains have been engineered to enable the specific study of phosphoinositide dynamics and functions in live cells. To examine PIP3 activity, PIP3-binding domains derived from Btk [151] and GRP1 [152] were used as indicators for PIP3. Fluorescent probes can be generated by creating fusion proteins, made up of PIP3-binding domains and fluorescent proteins [such as green fluorescent protein (GFP)]. These probes can then be expressed in living cells. The probes are evenly distributed in the cytosol. In response to different conditions or stimuli, the probes translocate to the cellular membrane where PIP3 is generated. Based on the extent of probe accumulation, we can identify the level of PIP3 production. The same strategy has been applied to PI(4,5)P2 using a PH domain from PLCγ [63, 153]. However, several considerations must be taken into account when interpreting imaging

Endosome Synaptic vesicle

Post synatptic density PI(4)P PI(3,5)P2 PIP3 PI(4,5)P2 PI(3,4)P2 PI(3)P PI(5)P AMPAR NMDAR Glutamate

Early endosome

Late endosome

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experiments. As phosphoinositide-imaging experiments are typically performed in live cells, any alterations in cell shape or encounters with uneven membrane structures (e.g., membrane ruffles) can significantly affect the fluorescence intensity measurements, potentially leading to the generation of artifacts. Moreover, it can often be difficult to distinguish which membranous compartments the fluorescent fusion proteins translocated to. In order to overcome these limitations, we have developed Förster resonance energy transfer (FRET)based lipid probes for PIP3 [154] and DAG [155]. Our PIP3 FRET probe was constructed as follows (Fig. 3a): to enable specific PIP3 binding, a PH domain from GRP1 was introduced between cyan fluorescent protein (CFP) and a yellow fluorescent protein (YFP) variant, through rigid α-helical linkers consisting of repeated EAAAR sequences. We introduced a single di-glycine motif within one of the rigid linkers, which acts as a hinge and provided flexibility to the probe. A CAAX sequence from N-Ras was also attached to the probe to

Fig. 3 PIP3 probe and fluorescence lifetime imaging of PIP3 in spines. a PIP3 is produced PI3K, and then PIP3 on the plasma membrane can interact with the PH domain of the probe. This interaction induces a conformation change in the probe leading to an increase in FRET. b Images of fluorescence lifetimebased PIP3 probe (FLIMPA3) and FLIMPA mutant (mut), which is not able to bind to PIP3, were expressed in CA1 pyramidal neurons in hippocampal slices. The FLIMPA3 probe demonstrated that PIP3 is enriched at spines (compared to dendrites), but this enrichment was not observed in neurons expressing the FLIMPA mutants

assist plasma membrane targeting. The binding of PIP3 to the PH domain leads to a substantial change in the conformation of the probe. This flip-flop-type conformational change results in increased FRET from CFP to YFP and allows stable observation of PIP3 by dual-emission ratiometric imaging. Ratiometric imaging can cancel out the artifact derived from fluorescent intensity changes caused by membrane ruffling or changes in cell shape. There are two main strengths to this probe. Firstly, by using specific membrane target sequences, it enables the accurate examination of PIP3 dynamics at the membrane of interest. Secondly, the binding domain can be easily modified to enable the study of other phosphoinositides such as PI(4,5)P2, PI(4)P, and PI(3,4)P2 (as reported by Matsuda group) [19, 136, 156]. Additionally, for studies in thick tissue such as hippocampal slices or intact brains, we specifically engineered a fluorescence lifetime-based probe, which only considers the fluorescence lifetime of GFP and effectively eliminates any artifact caused by wavelength-

a PH YFP

CFP

Gly-Gly

αhelix (EAAAR)7 PIP3

PI3K activation Membrane Membrane target domain

b

High

Low PIP3

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dependent absorption that is often observed in thick tissue preparations [126].

Neuronal Functions of Phosphoinositides Excitatory synapses consist of two main components, a presynaptic terminal and a postsynaptic apposition. The presynaptic terminal is characterized by the presence of vesicles containing neurotransmitters [157]. The postsynaptic side is defined by the presence of micron-sized membrane protrusions, commonly known as spines. Within spines, there are several organelles and structures including the protein-rich structure called the postsynaptic density (PSD), where multiple interactions between different proteins, the smooth endoplasmic reticulum (SER), spine apparatus derived from SER, early and recycling endosomes, and lysosomes [157–159]. In addition to being the junctions of neurotransmission, synapses are also the elementary unit that can undergo synaptic plasticity, a phenomenon that underlies learning and memory [160]. Phosphoinositides are critically involved in regulating a wide range of synaptic functions including synaptic vesicle trafficking at the presynaptic side, and modulation of spine morphology and receptor exocytosis/endocytosis on the postsynaptic side [17, 126, 161, 162]. In this section, I will review the roles of phosphoinositides on modulating presynaptic and postsynaptic functions and plasticity mechanisms (for review, see ref. [58] for a comprehensive review on this topic).

Presynaptic Side The process of neurotransmitter release at nerve terminals consists of a series of exquisitely orchestrated steps [123, 163]. Firstly, synaptic vesicles need to be “primed” for neurotransmission. For this to occur, the synaptic vesicles bud from plasma membrane and then filled with neurotransmitter. The vesicles then translocate and dock at the active zone of the terminal, a place where proteins required for priming (such as Munc13, CAPS, and RIM) are present [92, 164, 165]. These docked and primed vesicles are at the penultimate stage of neurotransmitter release. However, neurotransmission can only occur upon arrival of an action potential, the ultimate trigger for vesicle fusion. Arrival of an action potential at the nerve terminal leads to the activation of voltage-gated Ca2+ channels, which causes a local and transient increase in intraterminal Ca2+. This elevation in Ca2+, subsequently, triggers the fusion of vesicles to the plasma membrane via the SNARE complex, resulting in neurotransmitter release. After fusion, the plasma membrane undergoes clathrin-mediated endocytosis, enabling the synaptic vesicle to be retrieved. (Since this review mainly focuses on phosphoinositide function in

synapses, please see refs. [64, 88] for further information on synaptic vesicle recycling.) Recent findings have demonstrated that the phosphoinositides have important functions in regulating presynaptic neurotransmission. PI(4,5)P2 is implicated in multiple stages of synaptic vesicle cycling including priming, fusion, and endocytosis [64, 166]. Priming requires the participation of a variety of proteins including Munc13, RIM, and CAPS [167]. Other proteins, such as ELKS and Piccolo/Bassoon, RIM-binding proteins, and Liprins, form a complex, which functions as a scaffold for synaptic vesicles at the active zone [168]. Interestingly, several proteins related to priming have PI(4,5)P2-binding domains (such as C2 and PTB domains) as shown in Table 1, potentially indicating that these proteins are regulated by PI(4,5)P2. In particular, the Südhof group showed that PI(4,5)P2 binds to the C2B domain of Munc13 in a Ca2+-dependent manner [169]. Introducing mutations into PI(4,5)P2 affected its binding ability to the C2B domain of Munc13, which lead to changes in neurotransmitter release. This indicated that PI(4,5)P2 controls synaptic vesicle exocytosis through the C2B domain of Munc13. In sum, this evidence suggests that PI(4,5)P2 plays an important role in the priming step through the binding of proteins necessary for priming. PI(4,5)P2 is also important for vesicle fusion. During fusion, PI(4,5)P2 works cooperatively with the synaptic vesicle Ca2+ sensor, synaptotagmin to regulate synaptic vesicle fusion. While the C2B domain of synaptotagmin binds to PIP3 in the absence of Ca2+, it subsequently binds to PI(4,5)P2 in the presence of Ca2+, suggesting that a lipid interaction switch occurs during depolarization [150]. The increase in Ca2+ triggers the association between the C2 domain and PI(4,5)P2. This leads to the insertion of the C2 domain into the plasma membrane, which promotes plasma membrane curvature and, subsequently, enhances the efficiency of vesicle fusion [99, 162]. After neurotransmitter release, synaptojanin dephosphorylates PI(4,5)P2 to generate PI(4)P during vesicle endocytosis. This reaction leads to the detachment of clathrin from the vesicle [41]. This evidence indicates that synaptic vesicles are enriched in PI(4)P and contain less PI(4,5)P2. The idea is supported by a study examining the subcellular distribution of PI(4,5)P2 in cultured hippocampal neurons using a PH domain–GFP probe [63]. Interestingly, a study revealed that FM4-64, a synaptic vesicle marker, and a PI(4,5)P2 probe were mutually exclusive at presynaptic boutons, suggesting a lack of significant amounts of PI(4,5)P2 on synaptic vesicles at quiescent synapses. Furthermore, the Chapman group recently showed using in vitro fusion assays with reconstituted SNARE proteins that while PI(4,5)P2 in vesicle membranes is not needed for vesicle fusion, PI(4,5)P2 in the target vesicle membrane is required [170]. Taken together, PI(4,5)P2 plays many important roles in the multistep process of synaptic vesicle cycling.

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Postsynaptic Side Spines are tiny membranous structures located on dendritic shafts. Spines can exhibit both functional and morphological plasticity in response to presynaptic stimulation and therefore serves as a flexible component within an otherwise fairly rigid neuronal network of the adult brain. Long-term potentiation (LTP) of synaptic transmission is thought to be an important mechanistic model that underlies memory formation [160]. The molecular mechanism for LTP at CA3–CA1 synapses can be explained as follows. The administration of high-frequency electrical stimulation to CA3 presynaptic neurons causes the release of neurotransmitters (including glutamate) from nerve terminals and, subsequently, leads to membrane depolarization of postsynaptic CA1 neurons. These events trigger the removal of Mg2+ from the channel pore of NMDAR, leading to NMDAR activation and further influx of Ca2+ through NMDAR. The elevation in Ca2+ can recruit AMPARs to the surface of spines from either extrasynaptic regions or intracellular compartments by the activation of signaling molecules, including Ca2+/calmodulin-dependent protein kinase II (CaMKII) [171]. The insertion of AMPARs at the postsynaptic membrane results in an increase in electrical transmission. Conversely, the application of low-frequency electrical stimulation decreases the electrical transmission, known as longterm depression (LTD). LTD causes the internalization of AMPARs from the membrane surface, a process mediated by calcineurin [172]. LTP and LTD are widely considered as possible models for the molecular events that underlie learning and memory [173]. Spines come in a variety of shapes and are typically classified into the following categories: mushroom, stubby, thin and filopodia [174]. Interestingly, the structure of spines is often deeply associated with synaptic plasticity [174] as well as neural diseases such as mental retardation [175]. Electron microscopy has revealed the presence of even finer structures on spines, including filopodia like protrusions, spinules, and spines perforations, all of which can dynamically change their structure in response to theta burst stimulation [176] and glutamate application [177]. Additionally, spine size and AMPAR current are positively correlated, indicating a strong relationship between structural and functional changes [178]. To date, phosphoinositides have been implicated in a broad spectrum of spine functions including current transmission, spine morphology and size, and trafficking of AMPAR and NMDAR containing vesicles.

Functional Plasticity The phosphoinositides have been implicated in regulating functional plasticity, with PIP3 being one of the most commonly studied phosphoinositides. The Francesconi group

showed that theta burst-induced LTP in hippocampal CA1 pyramidal neurons was abolished in the presence of a PI3K inhibitor [121]. In addition, the Wang group showed that PI3K is directly associated with AMPAR when glycine-induced specific activation of NMDAR occurs. This suggests that PI3K activates and produces PIP3 at close spatial proximity to AMPAR [124]. Furthermore, the Esteban group reported that PIP3 in spines is crucial for maintaining AMPAR clustering [122]. Their findings indicated that a decrease of PIP3 in spines causes the movement of AMPAR from the postsynaptic density towards the perisynaptic membrane within the spine, which leads to synaptic depression. In another paper, they investigated the effect of PTEN on LTP and LTD in CA1 pyramidal neuronal cells [179]. PTEN is a protein that can decrease PIP3 levels. Interestingly, overexpression of PTEN in neurons abolished basal AMPA current. Next, the effect of PTEN inhibitor on LTD was examined. Incubation of slices with a PTEN inhibitor abolished electrically induced LTD. In summary, the regulation of PIP3 levels by PI3K and PTEN signaling plays an important role in modulating LTP and LTD by influencing the movement of AMPAR in spines. PI(3,5)P2 is also involved in the trafficking of AMPAR to the plasma membrane on the postsynaptic side [67]. In hippocampal neurons, serum- and glucocorticoid-inducible kinase 3 (SGK3) mRNA is upregulated after NMDAR activation. Subsequently, SGK3 can phosphorylate PIKfyve at serine 318 to activate it. Activated PIKfyve then produces PI(3,5)P2, which activates Rab11 located on AMPARcontaining vesicles, leading to the trafficking of vesicles to the surface of postsynaptic spines. It was also shown in this report that injection of PI(3,5)P2 into Xenopus oocytes expressing GluA1 could amplify the GluA1 current. PI(3,4)P2 also plays an important role in functional plasticity. INPP4A is an enzyme responsible for degrading PI(3,4)P2 to PI(3)P. The accumulation of this lipid induces glutamate excitotoxicity in the central nervous system and exhibits involuntary movement and mortality in INPP4A−/ −mice [17]. This study also showed that PI(3,4)P2, but not PIP3 and PI(4,5)P2, can massively increase glutamateinduced excitotoxicity through the accumulation of NMDARs at synapses. There are several reports that PI(4,5)P2 is involved in LTD in hippocampal neurons. The Dell’Acqua group showed that protein kinase A kinase-anchoring protein (AKAP)79/150 plays a pivotal role in NMDAR-dependent LTD [153, 180]. AKAP79/150 is targeted to spines through an N-terminal basic region that binds PI(4,5)P2, F-actin, and cadherin and forms a complex with PKA and calcineurin (CaN)/protein phosphatase 2B (PP2B) at static state. NMDA stimulation activates PLC, which is followed by degradation of PI(4,5)P2. A decrease in PI(4,5)P2 removes AKAP79/150 and PKA from spines, leading to the inability of AMPARs to be phosphorylated by PKA, resulting in LTD. This data

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suggests that a decrease in PI(4,5)P2 in spines induces LTD. In contrast, the Kanaho group showed that an NMDA-triggered increase in PI(4,5)P2 can induce LTD [181]. Ca2+ elevation by NMDAR activation activates protein phosphatase 1 (PP1) and calcineurin, causing dephosphorylation of PI(4)P5Kγ661, a splice variant of PI(4)P5Kγ at spines. The dephosphorylated PIP5Kγ661 increases the affinity to AP-2. The association with AP-2 activates PIP5Kγ661, which then leads to PI(4,5)P2 production. The newly synthesized PI(4,5)P2 then recruits endocytic components, which result in the endocytosis of AMPAR. The difference in PI(4,5)P2 dynamics during LTD between the two studies can be explained by a difference in the localization of PI(4,5)P2. At first, PI(4,5)P2 breakdown by PLC after NMDAR activation occurs at the postsynaptic density. This removes AKAP79/150 and PKA from spines, leading to AMPAR dephosphorylation. The dephosphorylated AMPAR then moves to the active endocytic zone, where PI(4)P5Kγ661 produces PI(4,5)P2.

Structural Plasticity/Spine Morphology Spine morphology is regulated by actin polymerization and depolymerization [174, 182, 183]. Considering that PI(4,5)P2 binds to almost all of the actin-binding proteins, it is reasonable to assume that PI(4,5)P2 may play an important role in regulating spine morphology. The Inoue group investigated the effect of PI(4,5)P2 levels on actin dynamics using a brandnew technique, rapamycin-induced protein dimerization in culture cells [184]. When PI(4,5)P2 was increased by rapamycin-induced recruitment of PI(4)P5K to the plasma membrane, COS-7, HeLa, and HEK293 cells formed bundles of motile actin filaments known as actin comets. This evidence directly suggests that PI(4,5)P2 regulates actin dynamics. Additionally, recent studies showed that Preso, which interacts with PI(4,5)P2 through the FERM domain can regulate spine morphology and dendritic outgrowth [107, 108]. Furthermore, several studies showed that PI(4,5)P2-binding proteins can regulate spine morphology and number [70, 79, 81, 83, 86]. However, direct evidence that PI(4,5)P2 exclusively regulates spine morphology through the PI(4,5)P2binding domains is currently limited, because the PI(4,5)P2binding regions of cofilin, profilin, gelsolin, etc., are often shared with the actin-binding region [185–187]. This characteristic makes it more challenging to studying the implications of PI(4,5)P2 on spine morphology. As for other phosphoinositides, a loss of PI(3)P in hippocampal neurons from conditional VPS34 deletion mice was associated with a loss of spines, and ultimately neurodegeneration [142]. The development of the glutamate uncaging method in 2004 has made a huge advancement to our understanding of the molecular mechanisms that underlie structural LTP (sLTP)

[178]. After glutamate is locally applied to a spine of interest by photolysis of caged glutamate, the spine becomes around three to four times bigger than the basal spine size, approximately 1–2 min after stimulation. The spine size then reaches a plateau, before shrinking slightly, but still maintains a larger size (compared to the size before stimulation) even after several hours [178]. The difference in spine size before and after glutamate stimulation is often interpreted as a potential mechanism that may underlie learning and memory. To date, several key players for the regulation of sLTP have been revealed as follows [188]. Uncaged glutamate induces an increase in Ca2+ concentration through NMDAR [178]. The Ca2+ increase activates calmodulin/CaMKII [189], leading to the activation of small GTPases such as Ras, cdc42, and RhoA [190]. These signals, subsequently, converged in the LIM kinase/cofilin pathway to modify spine size through actin polymerization/ depolymerization [182]. Recently, we investigated the function of PIP3 in regulating the spine morphology of CA1 pyramidal neurons in the hippocampus [126]. We discovered that glutamate uncaging induced not only an enlargement of spines but also the formation of small filopodia-like protrusions, termed spinules. Interestingly, spinules were observed during the first 2 min after stimulation on 50 % of the spines. The number of spinules increased in the presence of a PTEN inhibitor, which increased PIP3. Conversely, the number of spinules decreased following treatment with a PI3K inhibitor. Together, these results indicate that PIP3 regulates spinule formation. In order to visualize PIP3 dynamics in spines, we developed a fluorescence lifetime-based PIP3 probe, FLIMPA3. The probe was based on a ratiometric PIP3 FRET probe, but the donor CFP and acceptor YFP molecules were exchanged to mEGFP and sREACh, respectively, to obtain the fluorescence lifetime signal. When FLIMPA3 was expressed in CA1 pyramidal neurons in hippocampal slices and observed using two-photon microscopy, it was found that PIP3 was more highly concentrated in spines, compared to dendrites (Fig. 3b). There are reports in which active PI3K, which is formed just after the association with AMPAR, mainly resides in spines, while PTEN is localized to the dendritic shaft of CA1 neuronal pyramidal cells [124, 125]. Therefore, PIP3 accumulation is determined by the localization of PTEN and active PI3K. Next, we observed PIP3 dynamics during sLTP. After glutamate uncaging, basal PIP3 in the spine was reduced inversely to the enlargement of the spine. Interestingly, the reduction of PIP3 after stimulation was highly correlated with relative PIP3 enrichment in spines (compared to the dendritic shaft) before the stimulation. Therefore, the reduction in PIP3 is primarily due to the addition of membrane from the dendritic shaft. Furthermore, while PIP3 decreases globally in spines during stimulation, we observed a specific accumulation of PIP3 in spinules. Overall, this data suggest that PIP3 regulates spinule formation on spines subjected to sLTP induction. Additionally, we found that PIP3 accumulates in spinules during sLTP.

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There are several studies investigating the properties of spinules using electron microscopy (EM). EM can highlight finer structures such as spinules and perforations on spines. Studies have shown that the number of spinules increases in response to theta burst stimulation [176], local glutamate stimulation [177], and high potassium application [191]. The length of spinules typically increase in response to exogenously applied glutamate [177]. EM studies have demonstrated that spinules could also be trans-synaptically endocytosed as separate vesicles by presynaptic terminals from the postsynaptic side [192]. Therefore, the spinules may serve as a mechanism for retrograde signaling or may aid postsynaptic membrane remodeling by removing excess membrane (Fig. 4) [192]. Alternatively, the McKinney group showed that the glutamate-induced extension of spines can lead to the formation of new synapses [177]. Compared to its postsynaptic role, the function of PIP3 at presynaptic terminals remains enigmatic. However, a recent Drosophila study showed that presynaptic PIP3 interacts with syntaxin 1A, a protein essential for vesicle fusion [123]. Modulation of PIP3 levels can change syntaxin 1A clustering and, subsequently, regulate neurotransmitter release. Furthermore, synaptojanin, another synaptic vesicle protein, was demonstrated to have higher affinity to PIP3 than PIP2 under basal conditions. Based on this evidence, the accumulation of PIP3 in spinules may influence both presynaptic and postsynaptic function. Fig. 4 Summary of the potential physiological significance of PIP3 accumulation in spinules. This figure outlines some of the possible biological functions of PIP3 in spinules. The stimulation of spines (a) can lead to the generation of spinules (b) on spines. Currently, there are three proposed roles for spinules. c-1 PIP3 signaling may occur at spinules to enable new synapses to form with functional presynaptic buttons [177]. c-2 PIP3 can be sent to the presynaptic site and may act as a messenger molecule for retrograde signaling [192]. c-3 The extended spinules may just be by-products of the process of spine reorganization [192]

Concluding Remarks Since the discovery of phosphoinositides, our knowledge of the function of different phosphoinositides species has greatly expanded. While the function of PI(4,5)P2 and PIP3 at synapses has been intensively studied, other synaptic phospholipids have been largely ignored. Interestingly, PI(4,5)P2, PIP2, PI(3,4)P2, and PI(3,5)P2 are involved in the regulation of proteins such as AMPAR and NMDAR, key components underlying synaptic plasticity. How do different phosphoinositide species and signaling pathways influence the dynamics and function of different proteins? One plausible explanation is the compartmentalized signaling of each phosphoinositide. In order to address the question further, information about subspine phosphoinositide dynamics is required. However, as the size of spines is very small, just ~1 μm, conventional light microscopy may not provide sufficient resolution to reliably study subspine activity as the resolution limit attained by light microscopy is close to the size of a spine. Super resolution fluorescent microscopy, such as STORM/PALM and STED, can provide a more detailed view of phosphoinositide function [193–196]. Another future direction is the relevance of phosphoinositides in disease states. Lithium has been an effective drug to improve bipolar disorder [197, 198]. The drug affects inositol monophosphatase (c)-1. New synapse formation

(c)-2. Retrograde signaling

PIP3 Low

(a)

High

(b)

Postsynaptic density Structural LTP stimulation

(c)-3. Spine reorganization

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and GSK3β, downstream signaling proteins of PIP3 [198, 199]. PTEN mutations cause Cowden disease, which is often accompanied by mental retardation, and are associated with an autism spectrum disorder [200]. The proteins related to PI(4,5)P2 generation and degradation, such as PI4K, PI(4)P5K, and synaptojanin, are related to bipolar disorder [201]. Synaptojanin 1 is also related to Alzheimer’s disease. Synj1 haplo-insufficient mice is protective against amyloidβ [202, 203] and induces its clearance [204]. Furthermore, there are several reports in which synj1 overexpression could be deleterious in Down syndrome [60, 205–208]. Acknowledgements I am grateful to L. Yu and F. Hullin-Matsuda for comments on the manuscript. I also thank the members of Hayashi’s laboratory for their support, critical reading of the manuscript and discussion. This work was supported by a Grant-in-Aid for Young Scientists (B) and the RIKEN Special Postdoctoral Researcher Program.

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