Molecular Mechanism of Hippocampal LTP: Learning and Memory (PDF)

Neuroscience Research 175 (2022) 3–15 Contents lists available at ScienceDirect Neuros...

Neuroscience Research 175 (2022) 3–15 Contents lists available at ScienceDirect Neuroscience Research journal homepage: www.elsevier.com/locate/neures Review article Molecular mechanism of hippocampal long-term potentiation – Towards multiscale understanding of learning and memory Yasunori Hayashi Department of Pharmacology, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan a r t i c l e i n f o a b s t r a c t Article history: Long-term potentiation (LTP) of synaptic transmission is considered to be a cellular counterpart of learn- Received 18 July 2021 ing and memory. Activation of postsynaptic NMDA type glutamate receptor (NMDA-R) induces trafficking Received in revised form 4 August 2021 of AMPA type glutamate receptors (AMPA-R) and other proteins to the synapse in sequential fashion. At Accepted 5 August 2021 the same time, the dendritic spine expands for long-term and modulation of actin underlies this (struc- Available online 8 August 2021 tural LTP or sLTP). How these changes persist despite constant diffusion and turnover of the component proteins have been the central focus of the current LTP research. Signaling triggered by Ca2+ -influx via Keywords: NMDA-R triggers kinase including Ca2+ /calmodulin-dependent protein kinase II (CaMKII). CaMKII can Synaptic plasticity Long-term potentiation sustain longer-term biochemical signaling by forming a reciprocally-activating kinase-effector complex Glutamate receptor with its substrate proteins including Tiam1, thereby regulating persistence of the downstream signal- Cytoskeleton ing. Furthermore, activated CaMKII can condense at the synapse through the mechanism of liquid-liquid Ca2+ /calmodulin-dependent protein kinase phase separation (LLPS). This increases the binding capacity at the synapse, thereby contributing to the II maintenance of enlarged protein complexes. It may also serve as the synapse tag, which captures newly Learning and memory synthesized proteins. © 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved. Contents 1. Introduction.............................................................................................................................................. 4 2. First step of LTP: induction............................................................................................................................... 4 3. Debate on pre- or postsynaptic expression and maintenance of LTP................................................................................... 5 4. AMPA-R trafficking....................................................................................................................................... 5 5. Structural LTP............................................................................................................................................ 6 6. Trafficking occurs in a wide range of proteins in a regulated fashion................................................................................... 7 7. Cellular signaling leading to LTP......................................................................................................................... 8 8. Possible substrates of CaMKII............................................................................................................................ 8 9. Cytoskeletal role of CaMKII.............................................................................................................................. 9 10. Reciprocal activation between CaMKII and effector proteins.......................................................................................... 9 11. Liquid-liquid phase separation of CaMKII as a novel mechanism of LTP.............................................................................. 9 12. Synaptic plasticity and human diseases............................................................................................................... 11 13. Concluding remarks................................................................................................................................... 11 Acknowledgments...................................................................................................................................... 12 References.............................................................................................................................................. 12 E-mail address: [email protected] https://doi.org/10.1016/j.neures.2021.08.001 0168-0102/© 2021 Elsevier B.V. and Japan Neuroscience Society. All rights reserved. Y. Hayashi Neuroscience Research 175 (2022) 3–15 1. Introduction stand the mechanism of learning and memory in the multiscale brain. In a book published in 1949, Donald Hebb described a model of how neurons in the brain store information (Hebb, 1949). He pro- 2. First step of LTP: induction posed that Ẅhen an axon of cell A is near enough to excite a cell B and repeatedly or persistently takes part in firing it, some growth LTP is often considered in three steps: “induction”, “expression” process or metabolic change takes place in one or both cells such and “maintenance”. The “induction” is a cellular signaling process that A’s efficiency, as one of the cells firing B, is increased¨. This that can be directly triggered by tetanic stimulation. The resultant property is now called Hebbian synaptic plasticity in honor of his signaling induces a change in the synapse that can be detected as an conceptual establishment. However, it took more than 20 years to increase in the synaptic transmission, which is called “expression”. experimentally demonstrate the Hebbian plasticity in an animal. Once transmission is enhanced, the “maintenance” mechanism per- Bliss and colleagues found in rabbit dentate gyrus that a brief strong petuates the status despite diffusion and turnover of component stimulation, or tetanic stimulation, of synaptic input (perforant molecules as well as any other processes that mediates reverse fibers) persistently potentiates the subsequent synaptic transmis- biochemical reactions, such as a phosphatase for a kinase reaction. sion, a phenomena widely known as long-term potentiation or LTP In the ’70 s and ’80 s, efforts were made to develop pharma- (Bliss and Collingridge, 1993; Bliss and Gardner-Medwin, 1973; cological reagents that selectively modulate glutamate-mediated Bliss and Lømo, 1973; Nicoll, 2017). The same phenomena can be synaptic transmission (Krogsgaard-Larsen and Hansen, 1992; also induced in vitro in hippocampal slice preparation, which facil- Watkins and Collingridge, 1989). This led to the identification of itated studies because of its accessibility (Alger and Teyler, 1976; two major classes of glutamate receptors in hippocampal synapses, Andersen et al., 1977; Lynch et al., 1976; Schwartzkroin and Wester, AMPA and NMDA-Receptors (AMPA-R and NMDA-R), based on 1975; Yamamoto and Chujo, 1978) (Fig. 1). The molecular mech- their specific agonists. These two types of receptors play differ- anism of LTP attracted the interest of a number of scientists. The ent roles. The basal transmission is mediated by AMPA-R but long-standing question in the field is what is the nature and identity not by NMDA-R (Collingridge et al., 1983; Muller et al., 1988). of “some growth process or metabolic change” that Hebb predicted. The tetanic stimulation activates NMDA-R and induces a tran- It can be rephrased as how a transient activation of a synapse trig- sient influx of Ca2+ into the postsynaptic compartment through the gers the persistent change in subsequent synaptic transmission receptor, which initiates the induction process leading to long-term efficacy. In this review, I start with the history of LTP research changes in AMPA-R, but not in NMDA-R (Kauer et al., 1990; Muller and then cover the recent results from my group as well as from et al., 1988). The postsynaptic Ca2+ is necessary and sufficient others. Finally, I will conclude with what is required to under- for the induction of LTP (Malenka et al., 1988, 1989), which trig- Fig. 1. Hippocampal long-term potentiation (LTP). A typical experiment of LTP recording from hippocampal slice is shown. A brief strong stimulation, for example tetanic stimulation at 100 Hz for one second, potentiates the subsequent synaptic transmission for long-term. In slice, it lasts for a few hours. In vivo, it can last days. 4 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 2. Induction and different possibilities of expression mechanism of LTP. Different models of molecular mechanism of LTP discussed in 90s. It was well accepted that during the induction of LTP, strong postsynaptic depolarization removes Mg2+ block of NMDA-R and let Ca2+ to flow into postsynaptic cytosol. This activates postsynaptic signaling molecules such as CaMKII (left). However, where it is expressed and maintained was long debated. While some argued an increase in presynaptic release probability, others insisted postsynaptic change, either by an increase in receptor activity or number (right). gers the induction process leading to the enhancement of AMPA-R which has been traditionally interpreted as a presynaptic increase transmission. in the probability of release, can also be attributed to a postsynaptic mechanism. If the site of persistent change is presynaptic, because the ini- 3. Debate on pre- or postsynaptic expression and tial induction of LTP requires postsynaptic NMDA-R activation maintenance of LTP and resulting Ca2+ influx, the postsynaptic side needs to some- how retrogradely communicate with the presynaptic side (Fig. 2). However, as to the site of expression and maintenance, there Several diffusible messengers have been proposed to play this was a great amount of confusion: whether it is presynaptic (an role including nitric oxide, carbon monoxide, arachidonic acid, increase in transmitter release) or postsynaptic (an increase in and platelet activating factor (Kato et al., 1994; O’Dell et al., postsynaptic sensitivity to glutamate) (Fig. 2). Using radiolabeled 1991; Williams et al., 1989; Zhuo et al., 1993). However, the glutamate, it was demonstrated that induction of LTP increases the reproducibility of such studies was still questioned (Selig et al., subsequent release of glutamate from hippocampal tissue (Dolphin 1996). Given these confusions, LTP studies obviously required a et al., 1982). The failure of synaptic transmission, interpreted as the breakthrough. failure in synaptic vesicle release, was decreased after the induc- tion of LTP, indicating a presynaptic increase in the probability of release (Stevens and Wang, 1994). The quantal analysis of the size of synaptic transmission also supports presynaptic expres- 4. AMPA-R trafficking sion (Bekkers and Stevens, 1990; Bolshakov and Siegelbaum, 1994; Malinow and Tsien, 1990). However, the same approach also led to The limitation of these studies was that they relied on elec- different conclusions, with some proposing mixed pre- and post- trophysiological recording and statistical analyses of the size of synaptic changes or purely postsynaptic changes (Edwards, 1991; synaptic response. The LTP studies required additional and inde- Kullmann and Nicoll, 1992; Larkman et al., 1992). pendent readout. The development of several key technologies Other approaches also supported postsynaptic change. A selec- revolutionized the study of LTP. First, neuronal gene introduc- tive increase in AMPA-R but not NMDA-R-mediated synaptic tion techniques such as transgenic technologies, virus vectors, and response is supportive of postsynaptic change rather than presy- chemical/physical methods allowed for specific molecular manipu- naptic change, which would affect both components equally (Foster lation (gain-of-function such as overexpression or loss-of-function and McNaughton, 1991; Kauer et al., 1988; Kullmann and Nicoll, such as knockdown) or labeling (Haas et al., 2002; Klein et al., 1992; 1992; Muller et al., 1988). An increase in responsiveness to exoge- Malinow et al., 2010; Pettit et al., 1995). Second, discovery of GFP nous glutamate (Davies et al., 1989) as well as an increase in the and its family of proteins allows for visualization of fine synaptic size of miniature excitatory postsynaptic current (mepsc) after structures, detection of molecular dynamics, and even biochemi- LTP (Manabe et al., 1992) are suggestive of postsynaptic change. cal reactions underlying synaptic plasticity (Giepmans et al., 2006; To add further complication, it turns out that a postsynaptically Malinow et al., 2010; Miyawaki, 2005; Shaner et al., 2005; Yasuda, silent synapse, a synapse which does not have functional AMPA- 2006). This is expedited by the employment of different fluores- R before the LTP induction, acquires AMPA-R response after the cence microscopy techniques, such as confocal, two-photon, and induction (unsilencing) (Isaac et al., 1995; Liao et al., 1995). This super-resolution microscopes (Choquet et al., 2021; Mainen et al., indicates that the decrease in the failure rate of synaptic response, 1999). 5 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 3. Differential synaptic transport of proteins after LTP induction. GluR1 subunit of AMPA-R, ␤-actin (A), and Homer1B (B) were each tagged with GFP (for AMPA-R, a pH sensitive GFP, super ecliptic pHluorine was used) and coexpressed with DsRed2 as a volume filler. LTP was induced by photo-uncaging of caged glutamate. Note that while GluR1 and actin were rapidly transported, Homer1B took one hour before it started accumulating at the synapse. From (Bosch et al., 2014). A silent synapse, a synapse which does not show any AMPA-R 5. Structural LTP response before LTP, can acquire response after LTP induction in an all-or-none fashion (Isaac et al., 1995; Liao et al., 1995). To explain Excitatory synapses in central excitatory neurons are typi- such all-or-none appearance of the AMPA-R response, it was pro- cally formed on dendritic spines, tiny mushroom-like protrusions posed that AMPA-Rs are translocated to the synapse upon induction on dendrites (Bosch and Hayashi, 2012; Harris and Kater, 1994; of LTP. To visualize this process, GFP-tagged AMPA-R was expressed Hayashi and Majewska, 2005; Yuste, 2010). A single dendritic spine in neurons in hippocampal slice culture (Bosch et al., 2014; Shi et al., typically harbors one synapse on its head and connects to the 1999). An LTP-inducing stimulation triggered the receptor traffick- dendritic shaft via a thin neck, thereby serving as a biochemical ing to the synapse (Bosch et al., 2014; Shi et al., 1999) (Fig. 3). and electrical compartment. During development, the number and Both lateral diffusion and exocytosis of the intracellular pool of structure of dendritic spines gradually become mature. Immature receptors take place in this process (Borgdorff and Choquet, 2002; neurons either do not have spines or have only filopodial structures Park et al., 2004; Passafaro et al., 2001; Patterson et al., 2010). lacking head (Hayashi and Majewska, 2005). It has been questioned Using a method to electrophysiologically tag exogenous AMPA-R, how dendritic spines change their shape during neuronal activity it was also demonstrated that induction of LTP triggers traffick- and LTP. ing of AMPA-R to the synapse (Hayashi et al., 2000). These studies Fifková et al. were the first to address this question. They ana- unambiguously showed that LTP is expressed and maintained post- lyzed the effect of tetanic stimulation on dendritic spine structure synaptically, although still did not rule out the involvement of the under an electron microscope in hippocampal dentate gyrus in presynaptic side. intact animal that underwent LTP induction similarly to Bliss and 6 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 4. Structure and regulation of CaMKII. (A) Domain structure of CaMKII subunits. The major phosphorylation sites are indicated by P. (B) 3D structure of the CaMKII␣ holoenzyme modeled from cryo-EM images and crystal structures. Color code is consistent with the left, based on PDB Accession 5U6Y (Myers et al., 2017). (C) Activation process of CaMKII. At basal status, the autoinhibitory domain inactivates the kinase domain. Ca2+ /CaM binding to the CaM-binding domain disinhibits the kinase. The autoinhibitory domain is phosphorylated, and the kinase becomes constitutively active in the absence of Ca2+. Lømo (1973). They found that dendritic spine starts expanding as 6. Trafficking occurs in a wide range of proteins in a early as 2 min, and lasted at least 23 h after tetanic stimulation regulated fashion (Fifková and Anderson, 1981; Fifková and Van Harreveld, 1977; Van Harreveld and Fifková, 1975). However, these observations were Extensive efforts have been made to identify the molecules made across different preparations in fixed tissue. involved in glutamatergic synaptic transmission. Both AMPA-R and Hosokawa et al. (1994) attempted a time-lapse imaging of den- NMDA-R subunits were cloned by employing a Xenopus oocyte dritic spine in live neurons under LTP using DiI-labeling and a cloning system and further by cross-hybridization (Hollmann et al., confocal microscope. They found an increase in the length of spines 1989; Moriyoshi et al., 1991; Traynelis et al., 2010). Proteins that 3 h after chemical induction of LTP. Later, in a neuron expressing directly and indirectly interact with the receptors were identi- GFP, which fills the cytosol and reveals fine protrusive structures fied using various methods such as yeast-two hybrid screening, of a neuron, a local tetanic stimulation generates new spines and coimmunoprecipitation, and mass spectrometric analysis (Sheng enlarges the existing spines (Engert and Bonhoeffer, 1999; Maletic- and Hoogenraad, 2007). These efforts of more than two decades Savatic et al., 1999; Malinow et al., 2010; Okamoto et al., 2004). identified literally thousands of molecules of all different cate- However, in these studies, it was difficult to confirm whether gories of cellular components, ranging from cell surface receptors the spine under observation underwent LTP or not, or if they and channels, scaffolding proteins, cytoskeletons, signal trans- were even stimulated, because presynaptic axons were not visi- duction machineries, protein synthesis, and membrane trafficking ble. This was resolved by the development of two-photon uncaging machineries. These proteins comprise an electron-dense structure of caged-glutamate, which releases glutamate upon illumination beneath the synapse called the postsynaptic density (PSD). with a two-photon laser. With this, it became possible to precisely When AMPA-R is trafficked and the dendritic spine expands, stimulate a single spine and observe the processes underlying LTP, how do these PSD proteins behave? Are they translocated to which could be also confirmed by electrophysiological recording the synapse together with AMPA-R or not? By using two-photon (Matsuzaki et al., 2001, 2004). This confirms that LTP and synaptic uncaging of caged-glutamate and GFP-fusion proteins, Bosch et al. trafficking of AMPA-R are accompanied by an expansion of den- (2014) systematically tested the translocation of different postsyn- dritic spines, which I call here structural LTP or sLTP (Fig. 3). Like aptic proteins (Fig. 3). They found that the translocation of proteins electrically induced and recorded LTP, sLTP is also persistent, last- occurs in an ordered fashion. First, actin and its regulators are ing at least a few hours (Bosch et al., 2014; Kwon and Sabatini, translocated within a few minutes in parallel with the spine expan- 2011; Matsuzaki et al., 2004; Patterson et al., 2010). sLTP is specific sion (Fig. 3A). By using Förster resonance energy transfer (FRET) to the stimulated spine and requires NMDA-R activation, reca- imaging, it was found that actin is rapidly translocated within 20 s pitulating the properties of LTP. Therefore, it is highly likely that after LTP induction, which serves as a driving force to expand the these two phenomena are mechanistically linked. By also using dendritic spine (Okamoto et al., 2004). Then AMPA-R, actin bind- glutamate uncaging, correlated enlargement of presynaptic bou- ing proteins and kinases are translocated in a manner following the tons was observed after sLTP induction (Meyer et al., 2014). The expansion of the dendritic spine. Finally, PSD scaffolding proteins presynaptic boutons expand, but much more slowly compared such as Homer1B and Shank are translocated after one hour, in a with dendritic spines. The boutons took three hours to gradually manner requiring protein synthesis (Fig. 3B). This delayed translo- expand whereas the spine immediately (∼1 min) enlarges after cation of PSD scaffolding proteins may be a mechanism of late stimulation. So, there is a disparity between the two sides of the phase LTP (L-LTP), which shares the requirement of protein synthe- synapse in the early phase of LTP, which is balanced at the later sis (Bosch et al., 2014; Meyer et al., 2014; Panja and Bramham, 2014; stage. Pinho et al., 2020) (Fig. 3B). This stepwise translocation of differ- 7 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 5. Activation of CaMKII upon LTP induction observed by FRET. CaMKII activity is transiently enhanced by stimulation by glutamate uncaging, but then quickly returned to the baseline levels. Note that blue in the pseudocolor indicates activation; modified from (Saneyoshi et al., 2019). ent proteins explains the synaptic consolidation process where the Once this reaction takes place, the autoinhibitory domain no synapse becomes more tolerant to reversal by depotentiating stim- longer inhibits the catalytic domain, thereby making CaMKII ulation (prolonged, low frequency stimulation) after LTP induction constitutively active. over time. LTP can be more readily reversed by a depotentiation Different models have been proposed as to CaMKII’s regulation protocol if applied within a short time window after LTP induc- and involvement in the maintenance of LTP. An earlier model pro- tion when the synapse is still nascent (Fujii et al., 1991; Yang et al., posed that the constitutive, Ca2+ -independent activity of CaMKII 2008). As the potentiated state is gradually consolidated with the increases persistently after LTP induction, thereby maintaining the delayed translocation of proteins, synapses become more resistant enhanced synaptic transmission (Fukunaga et al., 1993; Lisman to depotentiation. et al., 2002). Lee et al. challenged this view by optically monitor- ing the CaMKII activity by using a FRET-FLIM sensor, Camui (Kwok et al., 2008; Takao et al., 2005), during sLTP. They found that the 7. Cellular signaling leading to LTP duration of CaMKII activation lasts only ∼1 min after the stimu- lation (Chang et al., 2017; Lee et al., 2009; Saneyoshi et al., 2019) Among literally hundreds of molecules implicated in LTP (Sanes (Fig. 5). The same group generated a photoactivatable inhibitor of and Lichtman, 1999), arguably, CaMKII is the most well-studied as a CaMKII and found that photoactivation of the inhibitor is effective signaling molecule mediating LTP (Hell, 2014; Lisman et al., 2012). in blocking the structural plasticity only during induction but not The rise in postsynaptic Ca2+ concentration triggers activation of during maintenance (Murakoshi et al., 2017). CaMKII. Postsynaptic inhibition of the kinase blocks LTP (Malenka However, the amount of total CaMKII at the synapse increases et al., 1989; Malinow et al., 1988, 1989). Consistently, genetic abla- after LTP induction persistently (Bosch et al., 2014; Shen and Meyer, tion of CaMKII impairs LTP as well as learning and memory (Hinds 1999). Lee et al. (2009) found that CaMKII activity returns to the et al., 1998; Silva et al., 1992). Also, introduction of the active form basal level but they did not confirm that the basal level of activity of CaMKII is sufficient to induce enhancement of synaptic trans- is null. Indeed, there is always basally activated CaMKII in unstimu- mission and synaptic insertion of AMPA-R (Hayashi et al., 2000; lated hippocampal tissue preparation (Fukunaga et al., 1993; Takao Lledo et al., 1995; Pettit et al., 1994; Poncer et al., 2002; Shirke et al., 2005). These indicate that there is a net increase in the amount and Malinow, 1997). Therefore, activity of CaMKII is necessary and of active CaMKII at each synapse after LTP induction. Therefore, a sufficient to induce LTP. consensus has not been yet met whether kinase activity is required CaMKII has an N-terminal kinase domain, an autoin- during the maintenance phase of LTP. hibitory/regulatory domain and a C-terminal association domain (Fig. 4A, B). In inactive CaMKII, the autoinhibitory domain masks the kinase domain. Ca2+ /calmodulin binding to the regulatory 8. Possible substrates of CaMKII domain unmasks the inhibition of the catalytic domain and activates the kinase (Fig. 4C). Once activated, CaMKII not only An obvious question is which CaMKII substrate is necessary for phosphorylates various substrates but also autophosphorylates LTP. More than 400 phosphorylation sites of CaMKII are reported itself at threonine (T) 286, located in the autoinhibitory domain (Phosphosite Plus, https://www.phosphosite.org), many of which (Hanson et al., 1989; Miller et al., 1988; Schworer et al., 1988). are relevant to the synaptic functions. To name a few, AMPA-R 8 Y. Hayashi Neuroscience Research 175 (2022) 3–15 subunit GluA1, NMDA-R subunits GluN2A and 2B, cell adhesion heterooligomers are the dominant subunits in the forebrain. They molecule Neuroligin, nitric oxide (NO) synthase, synaptic scaffold- share a basic structure except for the linker sequences between ing protein Homer, Shank, PSD-95, and SynGAP, and a transcription the autoinhibitory/regulatory region and the association domain. factor CREB, are among known substrates. The phosphorylation CaMKII␣ subunit has a short linker whereas ␤ subunit has a longer of AMPA-R is an obvious interest as a mechanism of LTP (Barria linker. Importantly, CaMKII␤ subunits can interact with filamen- et al., 1997b; Hayashi et al., 1997; McGlade-McCulloh et al., 1993; tous actin (F-actin) through this linker region (Lin and Redmond, Raymond et al., 1993). The GluA1 subunit of AMPA-R is phospho- 2009; O’Leary et al., 2006; Shen et al., 1998). Due to its oligomeric rylated by CaMKII at serine 831 (S831, numbering based on mature structure, a single CaMKII heteromer containing CaMKII␤ can inter- protein after signal peptide cleavage), which is proposed to increase act with more than one filament and bundle them together. With the channel conductance, thereby contributing the increased trans- this mechanism, CaMKII may serve as a structural element (Fink mission (Barria et al., 1997a; Derkach et al., 1999; Diering et al., et al., 2003; Okamoto et al., 2007). Consistently, down-regulation 2016; Roche et al., 1996). However, S831 does not conform to the of the CaMKII␤ subunit, but not ␣ subunit using shRNA converts known consensus sequence of CaMKII substrates, RXXS/T, and its the dendritic spine structure into a filopodial structure (Okamoto affinity towards CaMKII is significantly lower than other substrates et al., 2007). This phenotype could be rescued by overexpression of (Özden et al., 2020). Also, S831 phosphorylation is not required for CaMKII␤ with a kinase null mutation, indicating that the structural CaMKII-induced trafficking of AMPA-R (Hayashi et al., 2000). role of CaMKII␤ is independent of the kinase activity. Then how Hosokawa et al. used Phos-tag SDS-PAGE to detect the sto- does kinase activity affect the structural role? The linker region ichiometry of GluA1 phosphorylation (Hosokawa et al., 2015; is intrinsically disordered with multiple autophosphorylation sites Kinoshita et al., 2006). Phos-tag specifically interacts with phospho- and autophosphorylated CaMKII␤ dissociates from F-actin (Kim rylated residues and slows down the migration of phosphorylated et al., 2015). With these phosphorylations, the induction of LTP protein in SDS-PAGE compared with the unphosphorylated coun- unbundles F-actin, thereby generating a transient window for F- terpart. By blotting the sample with an antibody that equally actin remodeling by actin regulators such as Arp2/3 and cofilin detects both phosphorylated and unphosphorylated proteins, one (Kim et al., 2015). In this way, CaMKII stabilizes synaptic F-actin by can estimate the stoichiometry of the phosphorylation (Hosokawa bundling them together in a naïve synapse but allows for a transient et al., 2010). By using this method, Hosokawa et al. (2015) found time-window of actin modification after LTP-inducing stimulation. that the stoichiometry of S831 phosphorylation is less than 1% of total GluA1, even after induction of chemical LTP in dissociated 10. Reciprocal activation between CaMKII and effector culture. This is too low to explain the observed increase in synap- proteins tic transmission. Antibodies against phosphorylated AMPA-R at S831 and S845 (a protein kinase A phosphorylation site) have been Then what is the role of the ␣ subunit of CaMKII, which is more widely used to study the significance of the AMPA-R phosphoryla- abundant than ␤? CaMKII is known to interact with the intracel- tion (Mammen et al., 1997) but these antibodies are highly sensitive lular carboxyl tail of NMDA-R subunit GluN2B through a binding and ironically detected trace amount of phosphorylation, which pocket called the T-site, which is usually occupied by the autoin- may or may not be functional. On the other hand, a double knock- hibitory domain (Bayer et al., 2001). The same binding mode is in animal of S831A and S845A indeed shows impairment in LTP and shared by several other proteins including endogenous CaMKII long-term retention of memory (Lee et al., 2003). Because S831 and inhibitory peptide CaMKIIN, a Rac guanine nucleotide exchange S845 phosphorylation is higher in younger tissue (Hosokawa et al., factor (RacGEF) TIAM1, GJD2/connexin 36, LRRC7/densin-180, 2015), it might be due to a developmental effect. Therefore, phos- Drosophila EAG (ether-à-go-go or KCNH) voltage-dependent potas- phorylation of AMPA-R may occur in vivo and have a functional role sium channel, and small G-protein Rem2 (Alev et al., 2008; Bayer but not it is not the way initially thought. et al., 2001; Chao et al., 2010; Royer et al., 2018; Saneyoshi et al., 2019; Sun et al., 2004; Vest et al., 2007; Walikonis et al., 2001). This binding is triggered by Ca2+ /calmodulin and once 9. Cytoskeletal role of CaMKII bound, persists even after chelation of Ca2+ (Bayer et al., 2001; Hosokawa et al., 2021; Saneyoshi et al., 2019). While GluN2B CaMKII has been considered as a molecule that mediates Ca2+ and EAG potassium channels has canonical CaMKII phosphoryla- signaling. However, there are a few mysteries about CaMKII if it is tion site R-X-X-S/T sequence, others are pseudosubstrates, having purely a signaling molecule. First, CaMKII is highly abundant in the non-phosphorylatable residues instead of S/T (Özden et al., 2020). brain, especially at the synapse, comprising approximately 10–30% Interestingly, this binding locks CaMKII into an active conformation of the total protein in the hippocampal PSD fraction (Erondu and (Bayer et al., 2001; Saneyoshi et al., 2019). Tiam1, in turn, is phos- Kennedy, 1985). If CaMKII is a kinase involved in signaling, it is phorylated and activated by CaMKII, through this interaction. Thus, not required in such abundance, as a single molecule of CaMKII can CaMKII and Tiam1 form a reciprocally activating kinase-effector phosphorylate multiple substrate molecules. Second, CaMKII forms complex (RAKEC). The formation of RAKEC with Tiam1 is constitu- a dodecamer or tetradecamer in rotational symmetry through its tive, lasting at least 30 min after LTP induction. In this way, RAKEC association domain, as revealed by electron microscopic observa- may be a mechanism to maintain CaMKII activity and, at the same tion and X-ray crystallography (Hoelz et al., 2003; Kanaseki et al., time, specific downstream signaling thereby contributing to the 1991; Myers et al., 2017). The association domain shares structural maintenance of LTP. similarity with a protein of unknown function found in green algae and bacteria (McSpadden et al., 2019). During the evolution of ani- mals, the common ancestor of CaM family proteins (CaMKII as well 11. Liquid-liquid phase separation of CaMKII as a novel as CaMKI, CaMKIV, and CaMKK) fused with the association domain mechanism of LTP to form the unique structure of CaMKII, which is conserved in all living animal lineages. However, the functional significance of such In order to explore CaMKII-associated protein (CaMKIIAP) more oligomeric structure has not been made entirely clear. systematically, a pull-down experiment was carried out from brain CaMKII is encoded by four different genes CAMK2A-D, each tissue. The mass spectrometric analysis identified >100 proteins translated into CaMKII␣-␦ subunits (Tobimatsu and Fujisawa, (Baucum et al., 2015), though whether the binding involves the T- 1989). All subunits are expressed in the brain but CaMKII␣ and ␤ site or not is not tested in this study. I came to an idea that CaMKII 9 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 6. Liquid-liquid phase separation of CaMKII and GluN2B. CaMKII and GluN2B carboxyl terminus (GluN2Bc) were purified. GluN2Bc was labeled with a dimeric fluorescent protein to match the actual receptor stoichiometry. CaMKII, GluN2Bc, and calmodulin did not undergo liquid-liquid phase separation in the absence of Ca2+ , but they were condensed by the addition of Ca2+. The condensate persisted even after the chelation of Ca2+ with EGTA. This requires T286 phosphorylation because in the absence of ATP or by introducing the T286A mutation to CaMKII, the condensate did not persist after EGTA addition. From (Hosokawa et al., 2021). Fig. 7. Separation of AMPA-R and NMDA-R by CaMKII liquid-liquid phase separation. In the absence of Ca2+ , AMPA-R (represented by Stargazin (STGc), NMDA-R (represented by carboxyl tail of GluN2B subunit (GluN2Bc)), and PSD-95 are phase-separated, while CaMKII was present in the dilute phase. Upon addition of Ca2+ , STGc, and PSD-95 formed an in-phase phase and were separated from GluN2B and CaMKII. This persisted after the chelation of Ca2+ with EGTA From (Hosokawa et al., 2021). may serve as a crosslinker of the proteins thereby accumulating was added, they formed protein droplets (Fig. 6). Once formed, the them at the synapse. Especially, I was intrigued by a phenomenon of droplets persist even after the addition of EGTA, in a manner depen- liquid-liquid phase separation (LLPS) of biological macromolecules, dent on the autophosphorylation at T286. Therefore, CaMKII can where macromolecules such as RNA or proteins spontaneously con- remember the transient Ca2+ signal as a form of LLPS. Whether densate and form a protein droplet in solvent. The proteins that CaMKII undergoes LLPS in vivo is not fully demonstrated, but undergo LLPS often have multimeric structure, weak interaction, rapid and constant turnover of synaptically accumulated CaMKII and an intrinsically disordered region (Banani et al., 2017; Feng as revealed by fluorescence recovery after photobleaching assay et al., 2019; Hayashi et al., 2021; Hyman et al., 2014; Shin and (FRAP) or photoactivatable fluorescence protein is consistent with Brangwynne, 2017). The multimeric structure of CaMKII is indeed this view (Bosch et al., 2014; Lu et al., 2014; Okamoto et al., 2004). an ideal structure to undergo LLPS with multiple substrates and Addition of more proteins to the system revealed that acti- pseudosubstrate proteins, which themselves can be oligomers and vated CaMKII not only undergoes LLPS by itself but also defines cross-link them together. a postsynaptic nanodomain through this mechanism (Hosokawa Purified CaMKII and GluN2B carboxyl tail were mixed and et al., 2021). When AMPA-R (represented by an auxiliary subunit observed under microscope to test if they undergo LLPS. In the Stargazin), NMDA-R, and PSD-95 are present in the system, activa- absence of Ca2+ , these two proteins remained in the diluted phase tion of CaMKII partitioned AMPA-R and NMDA-R into two different and no condensate was observed. However, when Ca2+ /calmodulin phases (Hosokawa et al., 2021) (Fig. 7). Consistently at a synapse, 10 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 8. Segregation of AMPA-R and NMDA-R into nanodomain by CaMKII. Nanodomains of the AMPA and NMDA-Receptors were visualized by super-resolution microscopy dSTORM. Inhibition of the interaction between CaMKII and NMDA-Receptor GluN2B with CN21 reduced the separation of nanodomains. From (Hosokawa et al., 2021). SCR: scrambled peptide. CaMKII segregates AMPA-R and NMDA-R into different subsynaptic CaMKII, as well as synaptic adhesion molecules such as postsynap- nanodomains through this mechanism as revealed by superreso- tic neuroligin and its presynaptic counterpart, neurexin in human lution microscopy (Choquet et al., 2021; Hosokawa et al., 2021) patients with disorders such as schizophrenia, autism, intellectual (Fig. 8). The same mechanism places AMPA-R beneath the synap- abnormality, and attention deficit hyperactivity disorder (ADHD) tic vesicle release site, thereby forming a synaptic nanocolumn, a (Obi-Nagata et al., 2019; XiangWei et al., 2018). Of a particular vertical arrangement of presynaptic transmitter release site and interest to me is the F98S mutation of CaMKII ␣ subunit, which postsynaptic glutamate receptor (Hruska et al., 2018; Liu et al., causes intellectual disability in patients (Küry et al., 2017). F98 2021; Tang et al., 2016). Because AMPA-R is not saturated with glu- was identified by Bayer et al. as an amino acid residue CaMKII ␣ tamate at the synapse (Liu et al., 1999; Patneau and Mayer, 1990; subunit that constitutes the T-site and is important for binding to Tong and Jahr, 1994; Xie et al., 1997), this might serve as a novel GluN2B (Bayer et al., 2006). This mutation is expected to disrupts mechanism of LTP mediated by CaMKII (Fig. 9). LLPS, suggesting requirement of LLPS of CaMKII in normal cogni- Also, the formation of the condensed phase of CaMKII can serve tive functions. Not just genetic mutations, autoantibody against as a synapse tag, a conceptual binding site specifically formed in NMDA-R causes acute psychosis, especially young females carrying a synapse that underwent LTP for newly synthesized protein (Frey teratoma (Dalmau et al., 2007). Repeated stress in rodents reduces and Morris, 1997; Pinho et al., 2020; Redondo and Morris, 2011). A LTP and impairs AMPA-R trafficking which leads to memory deficit tag should be formed at a synapse without requiring protein syn- (Yuen et al., 2012). The expression of GluA1 subunit of AMPA-R thesis and serves as a binding site for newly synthesized proteins. and CaMKII are downregulated in patients with major depressive CaMKII can interact with multiple proteins through its T-site as disorder (Fuchsova et al., 2015; Tochigi et al., 2007; Duric et al., well as indirectly through F-actin, which also binds multiple pro- 2013). These studies indicate that the abnormalities in excitatory teins (Okamoto et al., 2009). Therefore, CaMKII condensed at the synaptic transmission and plasticity might cause a wide variety of synapse by LLPS mechanism may satisfy the long-sought identity neuropsychiatric and neurocognitive disorders and paved a way of a synapse tag. to development of novel therapeutic approaches towards these disorders. 12. Synaptic plasticity and human diseases 13. Concluding remarks It has been known that phencyclidine, an NMDA-R chan- nel blocker, induces a schizophrenic behavior in human (Javitt, Already in 1991, it was ridiculed that LTP is a long-term prob- 1987). Also, a rodent model of NMDA-R hypofunction exbibits lem (Edwards, 1991). In 1999, Sanes and Lichtman wrote a review an abnormal social behavior, similar to the negative symptoms titled “Can molecules explain long-term potentiation?” (Sanes and of schizophrenia (Mohn et al., 1999). Recent advances in exome Lichtman, 1999). This well represents the sentiment of the field at sequencing led to the discovery of genetic mutations in synap- that time with an endless debate on pre and post. However, it was tic proteins including NMDA-R and its binding partners including the year when LTP studies with novel approaches (Shi et al., 1999) 11 Y. Hayashi Neuroscience Research 175 (2022) 3–15 Fig. 9. Alignment of presynaptic release site and postsynaptic AMPA-R by CaMKII as a novel mechanism of synaptic plasticity. CaMKII segregates AMPA-R and NMDA-R into nanodomains. More AMPA-R are concentrated beneath the transmitter release site, resulting in more efficient synaptic response. This may be a new mechanism of synaptic plasticity. From (Hosokawa et al., 2021). started emerging and continues to this day. Indeed, it has been still HFSP Research GrantRGP0022/2013 and RGP0020/2019, and CREST a long-term journey, but I trust ongoing works will elucidate the JPMJCR20E4 from Japan Science and Technology Agency. mystery of memory, the biggest remaining question for mankind. Especially, a multiscale understanding from molecule to circuit is crucial. Elucidating when and where LTP takes place in living animal References during the formation and consolidation of memory will be a next major challenge of LTP studies. 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ACS Chem. Neurosci. 6, 615–631. all my past mentors, collaborators, and friends for their contribu- Bayer, K.U., De Koninck, P., Leonard, A.S., Hell, J.W., Schulman, H., 2001. Interaction tion on various aspects of the work described here. I also thank with the NMDA receptor locks CaMKII in an active conformation. Nature 411, 801–805. Emily Agnello for editing. This work was supported by Grant-in- Bayer, K.U., LeBel, E., McDonald, G.L., O’Leary, H., Schulman, H., De Koninck, P., 2006. Aid for Scientific ResearchJP21650080, JP16H01292, JP16H01438, Transition from reversible to persistent binding of CaMKII to postsynaptic sites JP16H02455, JP17K19631, JP18H05434, and JP19H01010 from and NR2B. J. Neurosci. 26, 1164–1174. the MEXT, Japan, The Uehara Memorial Foundation, The Naito Bekkers, J.M., Stevens, C.F., 1990. Presynaptic mechanism for long-term potentiation in the hippocampus. Nature 346, 724–729. 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