Enhancement of angiotensin II type 1 receptor-associated protein in the paraventricular nucleus suppresses angiotensin II-dependent hypertension.

The renin-angiotensin system in the brain plays a pivotal role in modulating sympathetic nerve activity and contributes to the pathogenesis of hypertension. Angiotensin II (Ang II) type 1 receptor (AT1R)-associated protein (ATRAP) promotes internalization of AT1R while suppressing pathological overactivation of AT1R signaling. However, the pathophysiological function of ATRAP in the brain remains unknown. Therefore, this study aims to investigate whether ATRAP in the paraventricular nucleus (PVN) is involved in neurogenic hypertension pathogenesis in Ang II-infused rats. The ATRAP/AT1R ratio, which serves as an indicator of tissue AT1R hyperactivity, tended to decrease within the PVN in the Ang II group than in the vehicle group. This suggests an Ang II-induced hyperactivation of the AT1R signaling pathway in the PVN. Lentiviral vectors were generated to stimulate ATRAP expression. At 6 weeks of age, rats were microinjected with LV-Venus (Venus-expressing lentivirus) or LV-ATRAP (Venus-ATRAP-expressing lentivirus). The rats were then randomly divided into four groups: (1) Vehicle/LV-Venus, (2) Vehicle/LV-ATRAP, (3) Ang II/LV-Venus, and (4) Ang II/LV-ATRAP. Two weeks after microinjection, vehicle or Ang II was administered systemically for 2 weeks. In the Ang II/LV-ATRAP group, systolic blood pressure at 1 and 2 weeks following administration was significantly lower than that in the Ang II/LV-Venus group. Furthermore, urinary adrenaline levels tended to decrease in the Ang II/LV-ATRAP group than in the Ang II/LV-Venus group. These findings suggest that enhanced ATRAP expression in the PVN suppresses Ang II-induced hypertension, potentially by suppressing hyperactivation of the tissue AT1R signaling pathway and, subsequently, sympathetic nerve activity.

Dynamics of AMPA receptors regulate epileptogenesis in patients with epilepsy.

The excitatory glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) contribute to epileptogenesis. Thirty patients with epilepsy and 31 healthy controls are scanned using positron emission tomography with our recently developed radiotracer for AMPARs, [11C]K-2, which measures the density of cell-surface AMPARs. In patients with focal-onset seizures, an increase in AMPAR trafficking augments the amplitude of abnormal gamma activity detected by electroencephalography. In contrast, patients with generalized-onset seizures exhibit a decrease in AMPARs coupled with increased amplitude of abnormal gamma activity. Patients with epilepsy had reduced AMPAR levels compared with healthy controls, and AMPARs are reduced in larger areas of the cortex in patients with generalized-onset seizures compared with those with focal-onset seizures. Thus, epileptic brain function can be regulated by the enhanced trafficking of AMPAR due to Hebbian plasticity with increased simultaneous neuronal firing and compensational downregulation of cell-surface AMPARs by the synaptic scaling.

Synthesis of [18F] fluorine-labeled K-2 derivatives as radiotracers for AMPA receptors.

INTRODUCTION: AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor) receptors play a central role in neurotransmission and neuronal function. A positron emission tomography (PET) tracer for AMPA receptors, [11C]K-2, was recently developed by us to visualize AMPA receptors in the living human brain. [11C]K-2 is a derivative of 4-[2-(phenylsulphonylamino)ethylthio]-2,6-difuluoro-phenoxyacetamide (PEPA), and is labeled with the radioactive isotope 11C, which has a short half-life. PET drugs are usually labeled with 18F because of its long half-life. Therefore, we screened and identified potential 18F-labeled PET drugs for AMPA receptors (AMPA-PET drugs), which could provide an image equivalent to that of [11C]K-2. METHODS: Derivatives of K-2 labeled with 18F were synthesized and administered to rats and PET imaging was performed. The transferability of each compound to the brain and its correlation with the PET image of [11C]K-2 were evaluated from the obtained PET images. Furthermore, the specific binding ability of promising compounds to the AMPA receptor was evaluated by the PET imaging of rats, which we specifically knocked down the expression of AMPA by the lentivirus-mediated introduction of short hairpin RNA (shRNA) targeted to subunits of the AMPA receptor (GluA1-A3). The specific binding ability was also evaluated through electrophysiological experiments with acute brain slices. RESULTS: Some of the synthesized 18F-labeled candidate compounds showed a distribution similar to that of K-2, with reasonable transferability to the brain. In addition, from the evaluation of the specific binding ability to the AMPA receptor, a promising structure of an 18F-labeled AMPA PET drug was identified. This study also revealed that the alkylation of the sulfonamide group of PEPA enhances brain transferability.

Epileptic discharges initiate from brain areas with elevated accumulation of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors.

Presurgical identification of the epileptogenic zone is a critical determinant of seizure control following surgical resection in epilepsy. Excitatory glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor is a major component of neurotransmission. Although elevated α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor levels are observed in surgically resected brain areas of patients with epilepsy, it remains unclear whether increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated currents initiate epileptic discharges. We have recently developed the first PET tracer for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor, [11C]K-2, to visualize and quantify the density of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors in living human brains. Here, we detected elevated [11C]K-2 uptake in the epileptogenic temporal lobe of patients with mesial temporal lobe epilepsy. Brain areas with high [11C]K-2 uptake are closely colocalized with the location of equivalent current dipoles estimated by magnetoencephalography or with seizure onset zones detected by intracranial electroencephalogram. These results suggest that epileptic discharges initiate from brain areas with increased α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors, providing a biological basis for epileptic discharges and an additional non-invasive option to identify the epileptogenic zone in patients with mesial temporal lobe epilepsy.

Biodistribution and radiation dosimetry of the positron emission tomography probe for AMPA receptor, [11C]K-2, in healthy human subjects.

[11C]K-2, a radiotracer exhibiting high affinity and selectivity for α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs), is suitable for the quantification of AMPARs in living human brains and potentially useful in the identification of epileptogenic foci in patients. This study aimed to estimate the radiation doses of [11C]K-2 in various organs and calculate the effective dose after injection of [11C]K-2 in healthy human subjects. Twelve healthy male subjects were registered and divided into two groups (370 or 555 MBq of [11C]K-2), followed by 2 h whole-body scans. We estimated the radiation dose of each organ and then calculated the effective dose for each subject. The highest uptake of [11C]K-2 was observed in the liver, while the brain also showed relatively high uptake. The urinary bladder exhibited the highest radiation dose. The kidneys and liver also showed high radiation doses after [11C]K-2 injections. The effective dose of [11C]K-2 ranged from 5.0 to 5.2 µSv/MBq. Our findings suggest that [11C]K-2 is safe in terms of the radiation dose and adverse effects. The injection of 370-555 MBq (10 to 15 mCi) for PET studies using this radiotracer is applicable in healthy human subjects and enables serial PET scans in a single subject.

Visualization of AMPA receptors in living human brain with positron emission tomography.

Although aberrations in the number and function of glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors are thought to underlie neuropsychiatric disorders, no methods are currently available for visualizing AMPA receptors in the living human brain. Here we developed a positron emission tomography (PET) tracer for AMPA receptors. A derivative of 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetamide radiolabeled with 11C ([11C]K-2) showed specific binding to AMPA receptors. Our clinical trial with healthy human participants confirmed reversible binding of [11C]K-2 in the brain according to Logan graphical analysis (UMIN000020975; study design: non-randomized, single arm; primary outcome: dynamics and distribution volumes of [11C]K-2 in the brain; secondary outcome: adverse events of [11C]K-2 during the 4-10 d following dosing; this trial met prespecified endpoints). In an exploratory clinical study including patients with epilepsy, we detected increased [11C]K-2 uptake in the epileptogenic focus of patients with mesial temporal lobe epilepsy, which was closely correlated with the local AMPA receptor protein distribution in surgical specimens from the same individuals (UMIN000025090; study design: non-randomized, single arm; primary outcome: correlation between [11C]K-2 uptake measured with PET before surgery and AMPA receptor protein density examined by biochemical study after surgery; secondary outcome: adverse events during the 7 d following PET scan; this trial met prespecified endpoints). Thus, [11C]K-2 is a potent PET tracer for AMPA receptors, potentially providing a tool to examine the involvement of AMPA receptors in neuropsychiatric disorders.

CRMP2-binding compound, edonerpic maleate, accelerates motor function recovery from brain damage.

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. No effective medication-mediated intervention to accelerate rehabilitation has been established. We found that a small compound, edonerpic maleate, facilitated experience-driven synaptic glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor delivery and resulted in the acceleration of motor function recovery after motor cortex cryoinjury in mice in a training-dependent manner through cortical reorganization. Edonerpic bound to collapsin-response-mediator-protein 2 (CRMP2) and failed to augment recovery in CRMP2-deficient mice. Edonerpic maleate enhanced motor function recovery from internal capsule hemorrhage in nonhuman primates. Thus, edonerpic maleate, a neural plasticity enhancer, could be a clinically potent small compound with which to accelerate rehabilitation after brain damage.

Social isolation suppresses actin dynamics and synaptic plasticity through ADF/cofilin inactivation in the developing rat barrel cortex.

Exposure to a stressful environment early in life can cause psychiatric disorders by disrupting circuit formation. Actin plays central roles in regulating neuronal structure and protein trafficking. We have recently reported that neonatal isolation inactivated ADF/cofilin, the actin depolymerizing factor, resulted in a reduced actin dynamics at spines and an attenuation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor delivery in the juvenile rat medial prefrontal cortex (mPFC), leading to altered social behaviours. Here, we investigated the impact of neonatal social isolation in the developing rat barrel cortex. Similar to the mPFC study, we detected an increase in stable actin fraction in spines and this resulted in a decreased synaptic AMPA receptor delivery. Thus, we conclude that early life social isolation affects multiple cortical areas with common molecular changes.

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex.

Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.

Sustained Enhancement of Lateral Inhibitory Circuit Maintains Cross Modal Cortical Reorganization.

Deprivation of one modality can lead to the improvement of other intact modalities. We have previously reported that visual deprivation drives AMPA receptors into synapses from layer4 to 2/3 in the barrel cortex and sharpens functional whisker-barrel map at layer2/3 2 days after the beginning of visual deprivation. Enhanced excitatory synaptic transmission at layer4-2/3 synapses is transient and returns to the base line level a week after the beginning of visual deprivation. Here we found that sharpened whisker-barrel function is maintained at least for a week in visually deprived animals. While increased AMPA receptor-mediated synaptic transmission at layer4-2/3 synapses dropped to the base line a week after the beginning of visual deprivation, lateral inhibitory synaptic transmission onto the neighboring barrel was kept strengthened for a week of visually deprived animals. Thus, transient strengthening of excitatory synapses at layer4-2/3 in the barrel cortex could trigger the enhancement of inhibitory inputs to neighboring barrel, and sustained lateral inhibition can maintain the sharpening of whisker-barrel map in visually deprived animals.

Nogo Receptor Signaling Restricts Adult Neural Plasticity by Limiting Synaptic AMPA Receptor Delivery.

Experience-dependent plasticity is limited in the adult brain, and its molecular and cellular mechanisms are poorly understood. Removal of the myelin-inhibiting signaling protein, Nogo receptor (NgR1), restores adult neural plasticity. Here we found that, in NgR1-deficient mice, whisker experience-driven synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) insertion in the barrel cortex, which is normally complete by 2 weeks after birth, lasts into adulthood. In vivo live imaging by two-photon microscopy revealed more AMPAR on the surface of spines in the adult barrel cortex of NgR1-deficient than on those of wild-type (WT) mice. Furthermore, we observed that whisker stimulation produced new spines in the adult barrel cortex of mutant but not WT mice, and that the newly synthesized spines contained surface AMPAR. These results suggest that Nogo signaling limits plasticity by restricting synaptic AMPAR delivery in coordination with anatomical plasticity.

Disrupted cortical function underlies behavior dysfunction due to social isolation.

Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.