Cell Biology of Canonical Wnt Signaling.

Wnt signaling has multiple functions beyond the transcriptional effects of ß-catenin stabilization. We review recent investigations that uncover new cell physiological effects through the regulation of Wnt receptor endocytosis, Wnt-induced stabilization of proteins (Wnt-STOP), macropinocytosis, increase in lysosomal activity, and metabolic changes. Many of these growth-promoting effects of canonical Wnt occur within minutes and are independent of new protein synthesis. A key element is the sequestration of glycogen synthase kinase 3 (GSK3) inside multivesicular bodies and lysosomes. Twenty percent of human proteins contain consecutive GSK3 phosphorylation motifs, which in the absence of Wnt can form phosphodegrons for polyubiquitination and proteasomal degradation. Wnt signaling by either the pharmacological inhibition of GSK3 or the loss of tumor-suppressor proteins, such as adenomatous polyposis coli (APC) and Axin1, increases lysosomal acidification, anabolic metabolites, and macropinocytosis, which is normally repressed by the GSK3-Axin1-APC destruction complex. The combination of these cell physiological effects drives cell growth.

A Balancing Act: Learning from the Past to Build a Future-Focused Opioid Strategy.

The harmful side effects of opioid drugs such as respiratory depression, tolerance, dependence, and abuse potential have limited the therapeutic utility of opioids for their entire clinical history. However, no previous attempt to develop effective pain drugs that substantially ameliorate these effects has succeeded, and the current opioid epidemic affirms that they are a greater hindrance to the field of pain management than ever. Recent attempts at new opioid development have sought to reduce these side effects by minimizing engagement of the regulatory protein arrestin-3 at the mu-opioid receptor, but there is significant controversy around this approach. Here, we discuss the ongoing effort to develop safer opioids and its relevant historical context. We propose a new model that reconciles results previously assumed to be in direct conflict to explain how different signaling profiles at the mu-opioid receptor contribute to opioid tolerance and dependence. Our goal is for this framework to inform the search for a new generation of lower liability opioid analgesics.

Gain of Additional BIRC3 Protein Functions through 3'-UTR-Mediated Protein Complex Formation.

Alternative 3' untranslated regions (3' UTRs) are widespread, but their functional roles are largely unknown. We investigated the function of the long BIRC3 3' UTR, which is upregulated in leukemia. The 3' UTR does not regulate BIRC3 protein localization or abundance but is required for CXCR4-mediated B cell migration. We established an experimental pipeline to study the mechanism of regulation and used mass spectrometry to identify BIRC3 protein interactors. In addition to 3'-UTR-independent interactors involved in known BIRC3 functions, we detected interactors that bind only to BIRC3 protein encoded from the mRNA with the long 3' UTR. They regulate several functions, including CXCR4 trafficking. We further identified RNA-binding proteins differentially bound to the alternative 3' UTRs and found that cooperative binding of Staufen and HuR mediates 3'-UTR-dependent complex formation. We show that the long 3' UTR is required for the formation of specific protein complexes that enable additional functions of BIRC3 protein beyond its 3'-UTR-independent functions.

JNK activity modulates postsynaptic scaffold protein SAP102 and kainate receptor dynamics in dendritic spines.

Synapse formation depends on the coordinated expression and regulation of scaffold proteins. The JNK family kinases play a role in scaffold protein regulation, but the nature of this functional interaction in dendritic spines requires further investigation. Here, using a combination of biochemical methods and live-cell imaging strategies, we show that the dynamics of the synaptic scaffold molecule SAP102 are negatively regulated by JNK inhibition, that SAP102 is a direct phosphorylation target of JNK3, and that SAP102 regulation by JNK is restricted to neurons that harbor mature synapses. We further demonstrate that SAP102 and JNK3 cooperate in the regulated trafficking of kainate receptors to the cell membrane. Specifically, we observe that SAP102, JNK3, and the kainate receptor subunit GluK2 exhibit overlapping expression at synaptic sites and that modulating JNK activity influences the surface expression of the kainate receptor subunit GluK2 in a neuronal context. We also show that SAP102 participates in this process in a JNK-dependent fashion. In summary, our data support a model in which JNK-mediated regulation of SAP102 influences the dynamic trafficking of kainate receptors to postsynaptic sites, and thus shed light on common pathophysiological mechanisms underlying the cognitive developmental defects associated with diverse mutations.

Pushing Forward the Future Tense: Perspectives of a Scientist.

This review is a somewhat chronological tale of my scientific life, emphasizing the why of the questions we asked in the lab and lessons learned that may be of value to nascent scientists. The reader will come to realize that the flow of my life has been driven by a combined life of the mind and life of the soul, intertwining like the strands of DNA.

Alternative splicing regulates adaptor protein binding, trafficking, and activity of the Vps10p domain receptor SorCS2 in neuronal development.

The Vps10p domain receptor SorCS2 is crucial for the development and function of the nervous system and essential for brain-derived neurotrophic factor (BDNF)-induced changes in neuronal morphology and plasticity. SorCS2 regulates the subcellular trafficking of the BDNF signaling receptor TrkB as well as selected neurotransmitter receptors in a manner that is dependent on the SorCS2 intracellular domain (ICD). However, the cellular machinery and adaptor protein (AP) interactions that regulate receptor trafficking via the SorCS2 ICD are unknown. We here identify four splice variants of human SorCS2 differing in the insertion of an acidic cluster motif and/or a serine residue within the ICD. We show that each variant undergoes posttranslational proteolytic processing into a one- or two-chain receptor, giving rise to eight protein isoforms, the expression of which differs between neuronal and nonneuronal tissues and is affected by cellular stressors. We found that the only variants without the serine were able to rescue BDNF-induced branching of SorCS2 knockout hippocampal neurons, while variants without the acidic cluster showed increased interactions with clathrin-associated APs AP-1, AP-2, and AP-3. Using yeast two-hybrid screens, we further discovered that all variants bound dynein light chain Tctex-type 3; however, only variants with an acidic cluster motif bound kinesin light chain 1. Accordingly, splice variants showed markedly different trafficking properties and localized to different subcellular compartments. Taken together, our findings demonstrate the existence of eight functional SorCS2 isoforms with differential capacity for interactions with cytosolic ligands dynein light chain Tctex-type 3 and kinesin light chain 1, which potentially allows cell-type specific SorCS2 trafficking and BDNF signaling.

Postsynaptic Density Proteins and Their Role in the Trafficking of Group I Metabotropic Glutamate Receptors.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system that regulates multiple different forms of synaptic plasticity, including learning and memory. Glutamate transduces its signal by activating ionotropic glutamate receptors and metabotropic glutamate receptors (mGluRs). Group I mGluRs belong to the G protein-coupled receptor (GPCR) family. Regulation of cell surface expression and trafficking of the glutamate receptors represents an important mechanism that assures proper transmission of information at the synapses. There is growing evidence implicating dysregulated glutamate receptor trafficking in the pathophysiology of several neuropsychiatric disorders. The postsynaptic density (PSD) region consists of many specialized proteins which are assembled beneath the postsynaptic membrane of dendritic spines. Many of these proteins interact with group I mGluRs and have essential roles in group I mGluR-mediated synaptic function and plasticity. This review provides up-to-date information on the molecular determinants regulating cell surface expression and trafficking of group I mGluRs and discusses the role of few of these PSD proteins in these processes. As substantial evidences link mGluR dysfunction and maladaptive functioning of many PSD proteins to the pathophysiology of various neuropsychiatric disorders, understanding the role of the PSD proteins in group I mGluR trafficking may provide opportunities for the development of novel therapeutics in multiple neuropsychiatric disorders.

STIM2 regulates NMDA receptor endocytosis that is induced by short-term NMDA receptor overactivation in cortical neurons.

Recent findings suggest an important role for the dysregulation of stromal interaction molecule (STIM) proteins, activators of store-operated Ca2+ channels, and the prolonged activation of N-methyl-D-aspartate receptors (NMDARs) in the development of neurodegenerative diseases. We previously demonstrated that STIM silencing increases Ca2+ influx through NMDAR and STIM-NMDAR2 complexes are present in neurons. However, the interplay between NMDAR subunits (GluN1, GluN2A, and GluN2B) and STIM1/STIM2 with regard to intracellular trafficking remains unknown. Here, we found that the activation of NMDAR endocytosis led to an increase in STIM2-GluN2A and STIM2-GluN2B interactions in primary cortical neurons. STIM1 appeared to migrate from synaptic to extrasynaptic sites. STIM2 silencing inhibited post-activation NMDAR translocation from the plasma membrane and synaptic spines and increased NMDAR currents. Our findings reveal a novel molecular mechanism by which STIM2 regulates NMDAR synaptic trafficking by promoting NMDAR endocytosis after receptor overactivation, which may suggest protection against excessive uncontrolled Ca2+ influx through NMDARs.

Novel Leptin-Based Therapeutic Strategies to Limit Synaptic Dysfunction in Alzheimer's Disease.

Accumulation of hyper-phosphorylated tau and amyloid beta (Aß) are key pathological hallmarks of Alzheimer's disease (AD). Increasing evidence indicates that in the early pre-clinical stages of AD, phosphorylation and build-up of tau drives impairments in hippocampal excitatory synaptic function, which ultimately leads to cognitive deficits. Consequently, limiting tau-related synaptic abnormalities may have beneficial effects in AD. There is now significant evidence that the hippocampus is an important brain target for the endocrine hormone leptin and that leptin has pro-cognitive properties, as activation of synaptic leptin receptors markedly influences higher cognitive processes including learning and memory. Clinical studies have identified a link between the circulating leptin levels and the risk of AD, such that AD risk is elevated when leptin levels fall outwith the physiological range. This has fuelled interest in targeting the leptin system therapeutically. Accumulating evidence supports this possibility, as numerous studies have shown that leptin has protective effects in a variety of models of AD. Recent findings have demonstrated that leptin has beneficial effects in the preclinical stages of AD, as leptin prevents the early synaptic impairments driven by tau protein and amyloid ß. Here we review recent findings that implicate the leptin system as a potential novel therapeutic target in AD.

Regulation of Mertk Surface Expression via ADAM17 and γ-Secretase Proteolytic Processing.

Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.

Embryonic stem cells are devoid of macropinocytosis, a trafficking pathway for activin A in differentiated cells.

Ligand-receptor complexes formed at the plasma membrane are internalised via various endocytic pathways that influence the ultimate signalling output by regulating the selection of interaction partners by the complex along the trafficking route. We report that, in differentiated cells, activin A-receptor complexes are internalised via clathrin-mediated endocytosis (CME) and macropinocytosis (MP), whereas in human embryonic stem cells (hESCs) internalisation occurs via CME. We further show that hESCs are devoid of MP, which becomes functional upon differentiation towards endothelial cells through mesoderm mediators. Our results reveal, for the first time, that MP is an internalisation route for activin A in differentiated cells, and that MP is not active in hESCs and is induced as cells differentiate.

The potential of intravenous topiramate for the treatment of status epilepticus.

There is considerable clinical evidence that topiramate (TPM) has a high potential in the treatment of refractory and super-refractory status epilepticus (RSE, SRSE). Because TPM is only approved for oral administration, it is applied as suspension via a nasogastric tube for SE treatment. However, this route of administration is impractical in an emergency setting and leads to variable absorption with unpredictable plasma levels and time to peak concentration. Thus, the development of an intravenous (i.v.) solution for TPM is highly desirable. Here we present data on two parenteral formulations of TPM that are currently being developed. One of these solutions is using sulfobutylether-ß-cyclodextrin (SBE-ß-CD; Captisol®) as an excipient. A 1% solution of TPM in 10% Captisol® has been reported to be well tolerated in safety studies in healthy volunteers and patients with epilepsy or migraine, but efficacy data are not available. The other solution uses the FDA- and EMA-approved excipient amino sugar meglumine. Meglumine is much more effective to dissolve TPM in water than Captisol®. A 1% solution of TPM can be achieved with 0.5-1% of meglumine. While the use of Captisol®-containing solutions is restricted in children and patients with renal impairment, such restrictions do not apply to meglumine. Recently, first-in-human data were reported for a meglumine-based solution of TPM, indicating safety and efficacy when used as a replacement for oral administration in a woman with epilepsy. Based on the multiple mechanisms of action of TPM that directly target the molecular neuronal alterations that are thought to underlie the loss of efficacy of benzodiazepines and other anti-seizure medications during prolonged SE and its rapid brain penetration after i.v. administration, we suggest that parenteral (i.v.) TPM is ideally suited for the treatment of RSE and SRSE. This paper was presented at the 8th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures held in September 2022.

Fifty years of research on status epilepticus: Seizures use hippocampal memory circuits to generate status epilepticus and disrupt brain development.

This is a review of my laboratory's interest in status epilepticus (SE), which spanned five decades. It started with a study of the role of brain mRNAs in memory, and with the use of electroconvulsive seizures to disrupt recently acquired memories. This led to biochemical studies of brain metabolism during seizures, and to the serendipitous development of the first model of self-sustaining SE. The profound inhibition of brain protein synthesis by seizures had implications for brain development, and we showed that severe seizures and SE in the absence of hypoxemia and other metabolic complications can disrupt brain and behavioral development, a concept that was not widely accepted at that time. We also showed that many experimental models of SE can cause neuronal death in the immature brain, even at very young ages. Our studies of self-sustaining SE showed that the transition from single seizures to SE is accompanied by internalization and transient inactivation of synaptic GABAA receptors, while extrasynaptic GABAA receptors are untouched. At the same time, NMDA and AMPA receptors move to the synaptic membrane, creating a "perfect storm" combining failure of inhibition and runaway excitation. Major maladaptive changes in protein kinases and neuropeptides, particularly galanin and tachykinins, also contribute to the maintenance of SE. The therapeutic implications of these results are that our current practice to start the treatment of SE with benzodiazepine monotherapy leaves the changes in glutamate receptors untreated and that sequential use of drugs gives seizures more time to aggravate changes in receptor trafficking. In experimental SE, we showed that drug combinations based on the receptor trafficking hypothesis are far superior to monotherapy in stopping SE late in its course. Combinations that include an NMDA receptor blocker such as ketamine are much better than combinations that follow current evidence-based guidelines, and simultaneous delivery of the drugs is far more effective than sequential delivery of the same drugs at the same dose. This paper was presented as a Keynote Lecture at the 8th London-Innsbruck Colloquium on Status Epilepticus and Acute Seizures held in September 2022.

Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice.

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.

Elevated Hippocampal CRMP5 Mediates Chronic Stress-Induced Cognitive Deficits by Disrupting Synaptic Plasticity, Hindering AMPAR Trafficking, and Triggering Cytokine Release.

Chronic stress is a critical risk factor for developing depression, which can impair cognitive function. However, the underlying mechanisms involved in chronic stress-induced cognitive deficits remain unclear. Emerging evidence suggests that collapsin response mediator proteins (CRMPs) are implicated in the pathogenesis of psychiatric-related disorders. Thus, the study aims to examine whether CRMPs modulate chronic stress-induced cognitive impairment. We used the chronic unpredictable stress (CUS) paradigm to mimic stressful life situations in C57BL/6 mice. In this study, we found that CUS-treated mice exhibited cognitive decline and increased hippocampal CRMP2 and CRMP5 expression. In contrast to CRMP2, CRMP5 levels strongly correlated with the severity of cognitive impairment. Decreasing hippocampal CRMP5 levels through shRNA injection rescued CUS-induced cognitive impairment, whereas increasing CRMP5 levels in control mice exacerbated memory decline after subthreshold stress treatment. Mechanistically, hippocampal CRMP5 suppression by regulating glucocorticoid receptor phosphorylation alleviates chronic stress-induced synaptic atrophy, disruption of AMPA receptor trafficking, and cytokine storms. Our findings show that hippocampal CRMP5 accumulation through GR activation disrupts synaptic plasticity, impedes AMPAR trafficking, and triggers cytokine release, thus playing a critical role in chronic stress-induced cognitive deficits.

GRASP1 ubiquitination regulates AMPA receptor surface expression and synaptic activity in cultured hippocampal neurons.

The synaptic expression of glutamate receptors of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) type is dynamically controlled by interaction with binding partners and auxiliary proteins. These proteins can be regulated by posttranslational modifications, including ubiquitination. In this work, we investigated the regulation of glutamate receptor interacting protein-associated protein 1 (GRASP1) by ubiquitin-dependent mechanisms and its impact on surface expression and activity of synaptic AMPA receptors. Cotransfection of GFP-ubiquitin decreased myc-GRASP1 protein levels in HEK293T cells, and this effect was inhibited upon transfection of an ubiquitin mutant that cannot be ubiquitinated on Lys48. In addition, transfection of cultured hippocampal neurons with GFP-ubiquitin reduced the dendritic levels of endogenous GRASP1 and decreased the surface expression of GluA1 AMPA receptor subunits, an effect that was partly reversed by cotransfection with GRASP1. Similarly, transfection of hippocampal neurons with GFP-ubiquitin decreased the amplitude of miniature excitatory postsynaptic currents (mEPSCs) mediated by Ca2+ -impermeable AMPA receptors, and this effect was abrogated by cotransfection of GRASP1. Together, the results show a role for ubiquitination in the regulation of the postsynaptic protein GRASP1, which has an impact on the surface distribution of AMPA receptors and on their activity at the synapse.

The combined effects of corticosterone and brain-derived neurotrophic factor on plasticity-related receptor phosphorylation and expression at the synaptic surface in male Sprague-Dawley rats.

Following acute exercise, a temporal window exists wherein neuroplasticity is thought to be heightened. Although a number of studies have established that pairing this post-exercise period with motor training enhances learning, the mechanisms through which exercise-induced priming occurs are not well understood. Previously, we characterized a rodent model of acute exercise that generates significant enhancement in glutamatergic receptor phosphorylation as a possible mechanism to explain how exercise-induced priming might occur. However, whether these changes are stimulated by peripheral factors (e.g., glucocorticoids), central effects (e.g., brain-derived neurotrophic factor (BDNF), or a combination of the two remains unclear. Herein, we explored the possible individual and/or cumulative contribution corticosterone (CORT) and BDNF may have on glutamate receptor phosphorylation and synaptic surface expression. Tissue slices from the sensorimotor cortex were prepared and acutely (30 min) incubated with either CORT (200 nM), BDNF (20 ng/mL), or the simultaneous application of CORT and BDNF (CORT+BDNF). Immunoblotting with biotinylated synaptoneurosomes (which provide an enrichment of proteins from the synaptic surface) suggested divergent effects between CORT and BDNF. Acute CORT application enhanced NMDA- (GluN2A, B) and AMPA- (GluA1) receptor phosphorylation, whereas BDNF preferentially increased synaptic surface expression of both NMDA- and AMPA-receptor subunits. The combined effects of CORT+BDNF resulted in a unique subset of signaling patterns that favored phosphorylation in the absence of surface expression. Taken together, these data provide a mechanistic framework for how CORT and BDNF may alter glutamatergic synapses during exercise-induced priming.

Epsins in vascular development, function and disease.

Epsins are a family of adaptor proteins involved in clathrin-dependent endocytosis. In the vasculature, epsins 1 and 2 are functionally redundant members of this family that are expressed in the endothelial cells of blood vessels and the lymphatic system throughout development and adulthood. These proteins contain a number of peptide motifs that allow them to interact with lipid moieties and a variety of proteins. These interactions facilitate the regulation of a wide range of cell signaling pathways. In this review, we focus on the involvement of epsins 1 and 2 in controlling vascular endothelial growth factor receptor signaling in angiogenesis and lymphangiogenesis. We also discuss the therapeutic implications of understanding the molecular mechanisms of epsin-mediated regulation in diseases such as atherosclerosis and diabetes.

Pharmacological rescue of Ras signaling, GluA1-dependent synaptic plasticity, and learning deficits in a fragile X model.

Fragile X syndrome, caused by the loss of Fmr1 gene function, is the most common form of inherited mental retardation, with no effective treatment. Using a tractable animal model, we investigated mechanisms of action of a few FDA-approved psychoactive drugs that modestly benefit the cognitive performance in fragile X patients. Here we report that compounds activating serotonin (5HT) subtype 2B receptors (5HT2B-Rs) or dopamine (DA) subtype 1-like receptors (D1-Rs) and/or those inhibiting 5HT2A-Rs or D2-Rs moderately enhance Ras-PI3K/PKB signaling input, GluA1-dependent synaptic plasticity, and learning in Fmr1 knockout mice. Unexpectedly, combinations of these 5HT and DA compounds at low doses synergistically stimulate Ras-PI3K/PKB signal transduction and GluA1-dependent synaptic plasticity and remarkably restore normal learning in Fmr1 knockout mice without causing anxiety-related side effects. These findings suggest that properly dosed and combined FDA-approved psychoactive drugs may effectively treat the cognitive impairment associated with fragile X syndrome.

Dopamine receptor D2 regulates GLUA1-containing AMPA receptor trafficking and central sensitization through the PI3K signaling pathway in a male rat model of chronic migraine.

BACKGROUND: The pathogenesis of chronic migraine remains unresolved. Recent studies have affirmed the contribution of GLUA1-containing AMPA receptors to chronic migraine. The dopamine D2 receptor, a member of G protein-coupled receptor superfamily, has been proven to have an analgesic effect on pathological headaches. The present work investigated the exact role of the dopamine D2 receptor in chronic migraine and its effect on GLUA1-containing AMPA receptor trafficking. METHODS: A chronic migraine model was established by repeated inflammatory soup stimulation. Mechanical, periorbital, and thermal pain thresholds were assessed by the application of von Frey filaments and radiant heat. The mRNA and protein expression levels of the dopamine D2 receptor were analyzed by qRT‒PCR and western blotting. Colocalization of the dopamine D2 receptor and the GLUA1-containing AMPAR was observed by immunofluorescence. A dopamine D2 receptor agonist (quinpirole) and antagonist (sulpiride), a PI3K inhibitor (LY294002), a PI3K pathway agonist (740YP), and a GLUA1-containing AMPAR antagonist (NASPM) were administered to confirm the effects of the dopamine D2 receptor, the PI3K pathway and GULA1 on central sensitization and the GLUA1-containing AMPAR trafficking. Transmission electron microscopy and Golgi-Cox staining were applied to assess the impact of the dopamine D2 receptor and PI3K pathway on synaptic morphology. Fluo-4-AM was used to clarify the role of the dopamine D2 receptor and PI3K signaling on neuronal calcium influx. The Src family kinase (SFK) inhibitor PP2 was used to explore the effect of Src kinase on GLUA1-containing AMPAR trafficking and the PI3K signaling pathway. RESULTS: Inflammatory soup stimulation significantly reduced pain thresholds in rats, accompanied by an increase in PI3K-P110ß subunit expression, loss of dopamine receptor D2 expression, and enhanced GLUA1-containing AMPA receptor trafficking in the trigeminal nucleus caudalis (TNC). The dopamine D2 receptor colocalized with the GLUA1-containing AMPA receptor in the TNC; quinpirole, LY294002, and NASPM alleviated pain hypersensitivity and reduced GLUA1-containing AMPA receptor trafficking in chronic migraine rats. Sulpiride aggravated pain hypersensitivity and enhanced GLUA1 trafficking in CM rats. Importantly, the anti-injury and central sensitization-mitigating effects of quinpirole were reversed by 740YP. Both quinpirole and LY294002 inhibited calcium influx to neurons and modulated the synaptic morphology in the TNC. Additional results suggested that DRD2 may regulate PI3K signaling through Src family kinases. CONCLUSION: Modulation of GLUA1-containing AMPA receptor trafficking and central sensitization by the dopamine D2 receptor via the PI3K signaling pathway may contribute to the pathogenesis of chronic migraine in rats, and the dopamine D2 receptor could be a valuable candidate for chronic migraine treatment.