Yasuhiro Funahashi

Structural plasticity of dendritic spines in the nucleus accumbens (NAc) is crucial for learning from aversive experiences. Activation of NMDA receptors (NMDARs) stimulates Ca2+-dependent signaling that leads to changes in the actin cytoskeleton, mediated by the Rho family of GTPases, resulting in postsynaptic remodeling essential for learning. We investigated how phosphorylation events downstream of NMDAR activation drive the changes in synaptic morphology that underlie aversive learning. Large-scale phosphoproteomic analyses of protein kinase targets in mouse striatal/accumbal slices revealed that NMDAR activation resulted in the phosphorylation of 194 proteins, including RhoA regulators such as ARHGEF2 and ARHGAP21. Phosphorylation of ARHGEF2 by the Ca2+-dependent protein kinase CaMKII enhanced its RhoGEF activity, thereby activating RhoA and its downstream effector Rho-associated kinase (ROCK/Rho-kinase). Further phosphoproteomic analysis identified 221 ROCK targets, including the postsynaptic scaffolding protein SHANK3, which is crucial for its interaction with NMDARs and other postsynaptic scaffolding proteins. ROCK-mediated phosphorylation of SHANK3 in the NAc was essential for spine growth and aversive learning. These findings demonstrate that NMDAR activation initiates a phosphorylation cascade crucial for learning and memory.

Protein phosphorylation, a key regulator of cellular processes, plays a central role in brain function and is implicated in neurological disorders. Information on protein phosphorylation is expected to be a clue for understanding various neuropsychiatric disorders and developing therapeutic strategies. Nonetheless, existing databases lack a specific focus on phosphorylation events in the brain, which are crucial for investigating the downstream pathway regulated by neurotransmitters. To overcome the gap, we have developed a web-based database named “Kinase-Associated Neural PHOspho-Signaling (KANPHOS).” This paper presents the design concept, detailed features, and a series of improvements for KANPHOS. KANPHOS is designed to support data-driven research by fulfilling three key objectives: (1) enabling the search for protein kinases and their substrates related to extracellular signals or diseases; (2) facilitating a consolidated search for information encompassing phosphorylated substrate genes, proteins, mutant mice, diseases, and more; and (3) offering integrated functionalities to support pathway and network analysis. KANPHOS is also equipped with API functionality to interact with external databases and analysis tools, enhancing its utility in data-driven investigations. Those key features represent a critical step toward unraveling the complex landscape of protein phosphorylation in the brain, with implications for elucidating the molecular mechanisms underlying neurological disorders. KANPHOS is freely accessible to all researchers at https://kanphos.jp.

INTRODUCTION: Since the emergence of the cholinergic hypothesis of Alzheimer's disease (AD), acetylcholine has been viewed as a mediator of learning and memory. Donepezil improves AD-associated learning deficits and memory loss by recovering brain acetylcholine levels. However, it is associated with side effects due to global activation of acetylcholine receptors. Muscarinic acetylcholine receptor M1 (M1R), a key mediator of learning and memory, has been an alternative target. The importance of targeting a specific pathway downstream of M1R has recently been recognized. Elucidating signaling pathways beyond M1R that lead to learning and memory holds important clues for AD therapeutic strategies. AREAS COVERED: This review first summarizes the role of acetylcholine in aversive learning, one of the outputs used for preliminary AD drug screening. It then describes the phosphoproteomic approach focused on identifying acetylcholine intracellular signaling pathways leading to aversive learning. Finally, the intracellular mechanism of donepezil and its effect on learning and memory is discussed. EXPERT OPINION: The elucidation of signaling pathways beyond M1R by phosphoproteomic approach offers a platform for understanding the intracellular mechanism of AD drugs and for developing AD therapeutic strategies. Clarifying the molecular mechanism that links the identified acetylcholine signaling to AD pathophysiology will advance the development of AD therapeutic strategies.

The N-methyl-D-aspartate receptor (NMDAR)-mediated structural plasticity of dendritic spines plays an important role in synaptic transmission in the brain during learning and memory formation. The Rho family of small GTPase RhoA and its downstream effector Rho-kinase/ROCK are considered as one of the major regulators of synaptic plasticity and dendritic spine formation, including long-term potentiation (LTP). However, the mechanism by which Rho-kinase regulates synaptic plasticity is not yet fully understood. Here, we found that Rho-kinase directly phosphorylated discs large MAGUK scaffold protein 2 (DLG2/PSD-93), a major postsynaptic scaffold protein that connects postsynaptic proteins with NMDARs; an ionotropic glutamate receptor, which plays a critical role in synaptic plasticity. Stimulation of striatal slices with an NMDAR agonist induced Rho-kinase-mediated phosphorylation of PSD-93 at Thr612. We also identified PSD-93-interacting proteins, including DLG4 (PSD-95), NMDARs, synaptic Ras GTPase-activating protein 1 (SynGAP1), ADAM metallopeptidase domain 22 (ADAM22), and leucine-rich glioma-inactivated 1 (LGI1), by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among them, Rho-kinase increased the binding of PSD-93 to PSD-95 and NMDARs. Furthermore, we found that chemical-LTP induced by glycine, which activates NMDARs, increased PSD-93 phosphorylation at Thr612, spine size, and PSD-93 colocalization with PSD-95, while these events were blocked by pretreatment with a Rho-kinase inhibitor. These results indicate that Rho-kinase phosphorylates PSD-93 downstream of NMDARs, and suggest that Rho-kinase mediated phosphorylation of PSD-93 increases the association with PSD-95 and NMDARs to regulate structural synaptic plasticity.

Dopamine regulates emotional behaviors, including rewarding and aversive behaviors, through the mesolimbic dopaminergic pathway, which projects dopamine neurons from the ventral tegmental area to the nucleus accumbens (NAc). Protein phosphorylation is critical for intracellular signaling pathways and physiological functions, which are regulated by neurotransmitters in the brain. Previous studies have demonstrated that dopamine stimulated the phosphorylation of intracellular substrates, such as receptors, ion channels, and transcription factors, to regulate neuronal excitability and synaptic plasticity through dopamine receptors. We also established a novel database called KANPHOS that provides information on phosphorylation signals downstream of monoamines identified by our kinase substrate screening methods, including dopamine, in addition to those reported in the literature. Recent advances in proteomics techniques have enabled us to clarify the mechanisms through which dopamine controls rewarding and aversive behaviors through signal pathways in the NAc. In this review, we discuss the intracellular phosphorylation signals regulated by dopamine in these two emotional behaviors.

Dysfunctional dopamine signaling is implicated in various neuropsychological disorders. Previously, we reported that dopamine increases D1 receptor (D1R)-expressing medium spiny neuron (MSN) excitability and firing rates in the nucleus accumbens (NAc) via the PKA/Rap1/ERK pathway to promote reward behavior. Here, the results show that the D1R agonist, SKF81297, inhibits KCNQ-mediated currents and increases D1R-MSN firing rates in murine NAc slices, which is abolished by ERK inhibition. In vitro ERK phosphorylates KCNQ2 at Ser414 and Ser476; in vivo, KCNQ2 is phosphorylated downstream of dopamine signaling in NAc slices. Conditional deletion of Kcnq2 in D1R-MSNs reduces the inhibitory effect of SKF81297 on KCNQ channel activity, while enhancing neuronal excitability and cocaine-induced reward behavior. These effects are restored by wild-type, but not phospho-deficient KCNQ2. Hence, D1R-ERK signaling controls MSN excitability via KCNQ2 phosphorylation to regulate reward behavior, making KCNQ2 a potential therapeutical target for psychiatric diseases with a dysfunctional reward circuit.

Protein phosphorylation plays critical roles in a variety of intracellular signaling pathways and physiological functions that are controlled by neurotransmitters and neuromodulators in the brain. Dysregulation of these signaling pathways has been implicated in neurodevelopmental disorders, including autism spectrum disorder, attention deficit hyperactivity disorder and schizophrenia. While recent advances in mass spectrometry-based proteomics have allowed us to identify approximately 280,000 phosphorylation sites, it remains largely unknown which sites are phosphorylated by which kinases. To overcome this issue, previously, we developed methods for comprehensive screening of the target substrates of given kinases, such as PKA and Rho-kinase, upon stimulation by extracellular signals and identified many candidate substrates for specific kinases and their phosphorylation sites. Here, we developed a novel online database to provide information about the phosphorylation signals identified by our methods, as well as those previously reported in the literature. The "KANPHOS" (Kinase-Associated Neural Phospho-Signaling) database and its web portal were built based on a next-generation XooNIps neuroinformatics tool. To explore the functionality of the KANPHOS database, we obtained phosphoproteomics data for adenosine-A2A-receptor signaling and its downstream MAPK-mediated signaling in the striatum/nucleus accumbens, registered them in KANPHOS, and analyzed the related pathways.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

Antipsychotics often cause tardive dyskinesia, an adverse symptom of involuntary hyperkinetic movements. Analysis of the US Food and Drug Administration Adverse Event Reporting System and JMDC insurance claims revealed that acetaminophen prevented the dyskinesia induced by dopamine D2 receptor antagonists. In vivo experiments further showed that a 21-day treatment with haloperidol increased the number of vacuous chewing movements (VCMs) in rats, an effect that was inhibited by oral acetaminophen treatment or intracerebroventricular injection of N-(4-hydroxyphenyl)-arachidonylamide (AM404), an acetaminophen metabolite that acts as an activator of the transient receptor potential vanilloid 1 (TRPV1). In mice, haloperidol-induced VCMs were also mitigated by treatment with AM404 applied to the dorsal striatum, an effect not seen in TRPV1-deficient mice. Acetaminophen prevented the haloperidol-induced decrease in the number of c-Fos+preproenkephalin+ striatal neurons in wild-type mice but not in TRPV1-deficient mice. Finally, chemogenetic stimulation of indirect pathway medium spiny neurons in the dorsal striatum decreased haloperidol-induced VCMs. These results suggest that acetaminophen activates the indirect pathway neurons by activating TRPV1 channels via AM404.

Neurons are highly polarized cells that have structurally and functionally distinct processes called axons and dendrites. How neurons establish polarity is one of the fundamental questions of neuroscience. In the last decade, significant progress has been made in identifying and understanding the molecular mechanisms responsible for neuronal polarization, primarily through researches conducted on cultured neurons. Advances in phosphoproteomics technologies and molecular tools have enabled comprehensive signal analysis and visualization and manipulation of signaling molecules for analyzing neuronal polarity. Furthermore, advances in gene transfer techniques have revealed the role of extracellular and intracellular signaling molecules in neuronal polarization in vivo. This review discusses the latest insights and techniques for the elucidation of the molecular mechanisms that control neuronal polarity.

Dopamine (DA) activates mitogen-activated protein kinase (MAPK) via protein kinase A (PKA)/Rap1 in medium spiny neurons (MSNs) expressing the dopamine D1 receptor (D1R) in the nucleus accumbens (NAc), thereby regulating reward-related behavior. However, how MAPK regulates reward-related learning and memory through gene expression is poorly understood. Here, to identify the relevant transcriptional factors, we perform proteomic analysis using affinity beads coated with cyclic AMP response element binding protein (CREB)-binding protein (CBP), a transcriptional coactivator involved in reward-related behavior. We identify more than 400 CBP-interacting proteins, including Neuronal Per Arnt Sim domain protein 4 (Npas4). We find that MAPK phosphorylates Npas4 downstream of PKA, increasing the Npas4-CBP interaction and the transcriptional activity of Npas4 at the brain-derived neurotrophic factor (BDNF) promoter. The deletion of Npas4 in D1R-expressing MSNs impairs cocaine-induced place preference, which is rescued by Npas4-wild-type (WT), but not by a phospho-deficient Npas4 mutant. These observations suggest that MAPK phosphorylates Npas4 in D1R-MSNs and increases transcriptional activity to enhance reward-related learning and memory.

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.

Accumulating information on eukaryotic protein phosphorylation implies a large and complicated phospho-signalling network in various cellular processes. Although a large number of protein phosphorylation sites have been detected, their physiological consequences and the linkage between each phosphorylation site and the responsible protein kinase remain largely unexplored. To understand kinase-oriented phospho-signalling pathways, we have developed novel substrate screening technologies. In this review, we described the in vitro and in vivo screening methods named kinase-interacting substrate screening analysis and kinase-oriented substrate screening analysis, respectively.

Protein phosphorylation plays a critical role in the regulation of cellular function. Information on protein phosphorylation and the responsible kinases is important for understanding intracellular signaling. A method for in vivo screening of kinase substrates named KIOSS (kinase-oriented substrate screening) has been developed. This protocol provides a method that utilizes phosphoprotein-binding modules such as 14-3-3 protein, the pin1-WW domain, and the chek2-FHA domain as biological filters to successfully enrich phosphorylated proteins related to intracellular signaling rather than housekeeping and/or structural proteins. More than 1000 substrate candidates for PKA, PKC, MAPK, and Rho-kinase in HeLa cells, as well as phosphorylation downstream of D1R, NMDAR, adenosine A2a receptor, PKA, PKC, MAPK, and Rho-kinase in mouse brain slice cultures have been identified by this method. An online database named KANPHOS (Kinase-Associated Neural Phospho-Signaling) provides the phosphorylation signals identified by these studies, as well as those previously reported in the literature. © 2019 by John Wiley & Sons, Inc.

Medium spiny neurons (MSNs) expressing dopamine D1 receptor (D1R) or D2 receptor (D2R) are major components of the striatum. Stimulation of D1R activates protein kinase A (PKA) through Golf to increase neuronal activity, while D2R stimulation inhibits PKA through Gi. Adenosine A2A receptor (A2AR) coupled to Golf is highly expressed in D2R-MSNs within the striatum. However, how dopamine and adenosine co-operatively regulate PKA activity remains largely unknown. Here, we measured Rap1gap serine 563 phosphorylation to monitor PKA activity and examined dopamine and adenosine signals in MSNs. We found that a D1R agonist increased Rap1gap phosphorylation in striatal slices and in D1R-MSNs in vivo. A2AR agonist CGS21680 increased Rap1gap phosphorylation, and pretreatment with the D2R agonist quinpirole blocked this effect in striatal slices. D2R antagonist eticlopride increased Rap1gap phosphorylation in D2R-MSNs in vivo, and the effect of eticlopride was blocked by the pretreatment with the A2AR antagonist SCH58261. These results suggest that adenosine positively regulates PKA in D2R-MSNs through A2AR, while this effect is blocked by basal dopamine in vivo. Incorporating computational model analysis, we propose that the shift from D1R-MSNs to D2R-MSNs or vice versa appears to depend predominantly on a change in dopamine concentration.