Aß peptide enhances GluA1 internalization via lipid rafts in Alzheimer's-related hippocampal LTP dysfunction.

Amyloid ß (Aß) is a central contributor to neuronal damage and cognitive impairment in Alzheimer's disease (AD). Aß disrupts AMPA receptor-mediated synaptic plasticity, a key factor in early AD progression. Numerous studies propose that Aß oligomers hinder synaptic plasticity, particularly long-term potentiation (LTP), by disrupting GluA1 (encoded by GRIA1) function, although the precise mechanism remains unclear. In this study, we demonstrate that Aß mediates the accumulation of GM1 ganglioside in lipid raft domains of cultured cells, and GluA1 exhibits preferential localization in lipid rafts via direct binding to GM1. Aß enhances the raft localization of GluA1 by increasing GM1 in these areas. Additionally, chemical LTP stimulation induces lipid raft-dependent GluA1 internalization in Aß-treated neurons, resulting in reduced cell surface and postsynaptic expression of GluA1. Consistent with this, disrupting lipid rafts and GluA1 localization in rafts rescues Aß-mediated suppression of hippocampal LTP. These findings unveil a novel functional deficit in GluA1 trafficking induced by Aß, providing new insights into the mechanism underlying AD-associated cognitive dysfunction.

Temporal and quantitative analysis of the functional expression of Ca2+-permeable AMPA receptors during LTP.

In the present study, we attempted to temporally and quantitatively analyze the functional contributions of Ca2+-permeable (CP) α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) during long-term potentiation (LTP) expression using electrophysiological and pharmacological approaches. In hippocampal CA1 neurons, using 1-naphthyl acetyl spermine (NASPM), a CP-AMPAR antagonist, we began by demonstrating that NASPM-sensitive components, probably including the GluA1 homomer, functionally contributed to about 15% of AMPAR-mediated EPSC amplitude in basal conditions. Then, when NASPM was treated at different time points (3-30 min) after LTP induction, it was found that LTP was almost completely impaired at 3 or 10 min but maintained at 20 or 30 min, although its potentiation was reduced. Further temporal and quantitative analysis revealed that the functional expression of CP-AMPARs began increasing approximately 20 min after LTP induction and reached more than twice the basal level at 30 min. These results suggest that CP-AMPARs in the first 3-10 min of LTP might play an important role in LTP maintenance. Moreover, their decay time was also significantly increased at 30 min, suggesting that CP-AMPARs changed not only quantitatively in LTP but also qualitatively.

Monitoring the glycosylation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-type glutamate receptors using specific antibodies reveals a novel regulatory mechanism of N-glycosylation occupancy by molecular chaperones in mice.

In the mammalian nervous system, protein N-glycosylation plays an important role in neuronal physiology. In this study, we performed a comprehensive N-glycosylation analysis of mouse GluA1, one of the major subunits of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate type glutamate receptor, which possesses six potential N-glycosylation sites in the N-terminal domain. By mass spectrometry-based analysis, we identified the N-glycoforms and semiquantitatively determined the site-specific N-glycosylation occupancy of GluA1. In addition, only the N401-glycosylation site demonstrated incomplete N-glycosylation occupancy. Therefore, we generated a peptide antibody that specifically detects the N401-glycan-free form to precisely quantify N401-glycosylation occupancy. Using this antibody, we clarified that N401 occupancy varies between cell types and increases in an age-dependent manner in mouse forebrains. To address the regulatory mechanism of N401-glycosylation, binding proteins of GluA1 around the N401 site were screened. HSP70 family proteins, including Bip, were identified as candidates. Bip has been known as a molecular chaperone that plays a key role in protein folding in the ER (endoplasmic reticulum). To examine the involvement of Bip in N401-glycosylation, the effect of Bip over-expression on N401 occupancy was evaluated in HEK293T cells, and the results demonstrated Bip increases the N401 glycan-free form by mediating selective prolongation of its protein half-life. Taken together, we propose that the N401-glycosite of GluA1 receives a unique control of modification, and we also propose a novel N-glycosylation occupancy regulatory mechanism by Bip that might be associated with α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors function in the brain.

AMPA receptors in the synapse turnover by monomer diffusion.

The number and subunit compositions of AMPA receptors (AMPARs), hetero- or homotetramers composed of four subunits GluA1-4, in the synapse is carefully tuned to sustain basic synaptic activity. This enables stimulation-induced synaptic plasticity, which is central to learning and memory. The AMPAR tetramers have been widely believed to be stable from their formation in the endoplasmic reticulum until their proteolytic decomposition. However, by observing GluA1 and GluA2 at the level of single molecules, we find that the homo- and heterotetramers are metastable, instantaneously falling apart into monomers, dimers, or trimers (in 100 and 200 ms, respectively), which readily form tetramers again. In the dendritic plasma membrane, GluA1 and GluA2 monomers and dimers are far more mobile than tetramers and enter and exit from the synaptic regions. We conclude that AMPAR turnover by lateral diffusion, essential for sustaining synaptic function, is largely done by monomers of AMPAR subunits, rather than preformed tetramers.

N-glycosylation of the AMPA-type glutamate receptor regulates cell surface expression and tetramer formation affecting channel function.

The AMPA-type glutamate receptor (AMPA-R) plays a primary role in principal excitatory synaptic transmission and many neuronal functions including synaptic plasticity that underlie learning and memory. N-glycosylation is one of the major post-translational modifications of membrane proteins, but its specific roles in neurons remain largely unknown. AMPA-R subunits are N-glycosylated at their extracellular domains during their biosynthesis in the lumen of the endoplasmic reticulum and Golgi system. Six N-glycosylation sites are presumed to exist in the extracellular domain of GluA1, which is a member of the AMPA-R subunits. We observed that the intracellular trafficking and cell surface expression were strongly suppressed in the GluA1 mutants lacking N-glycans at N63/N363 in HEK293T cells. Multimer analysis using Blue Native-PAGE displayed the impaired tetramer formation in the glycosylation mutants (N63S and N363S), indicating that the mis-transport was caused by impaired tetramer formation. N63S and N363S mutants were primarily degraded via the lysosomal pathway. Flag-tagged N363S GluA1, but not N63S GluA1, expressed in primary cortical neuron cultures prepared from GluA1 knockout mice was observed to localize at the cell surface. Co-expression of GluA2 partially rescued tetramer formation and the cell surface expression of N363S GluA1 but not N63S GluA1, in HEK293T cells. Electrophysiological analysis also demonstrated functional heteromers of N363S GluA1 with GluA2. These data suggest that site-specific N-glycans on GluA1 subunit regulates tetramer formation, intracellular trafficking, and cell surface expression of AMPA-R. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Suppression of ciliary movements by a hypertonic stress in the newt olfactory receptor neuron.

Olfactory receptor neurons isolated from the newt maintain a high activity of the ciliary beat. A cilium of neuron is so unique that only little is known about regulatory factors for its beat frequency. We examined the olfactory receptor neuron immersed in various extracellular media under the video-enhanced differential interference contrast microscope. The activation of voltage-gated Ca2+ channels by K+ depolarization or by application of Ca2+ to membrane-permeabilized olfactory cells did not affect the ciliary movement, suggesting that Ca2+ influx through the cell membrane has no direct effect on the movement. However, when an extracellular medium contained NaCl or sucrose at concentrations only 30% higher than normal levels, ciliary movement was greatly and reversibly suppressed. In contrast, a hypotonic solution of such a solute did not change the ciliary movement. The hypertonic solutions had no effect when applied to permeabilized cells. Suction of the cell membrane with a patch pipette easily suppressed the ciliary movement in an isotonic medium. Application of positive pressure inside the cell through the same patch pipette eliminated the suppressive effect. From these findings, we concluded that the hypertonic stress suppressed the ciliary movement not by disabling the motor proteins, microtubules, or their associates in the cilia, but rather by modifying the chemical environment for the motor proteins. The ciliary motility of the olfactory receptor cell is directly sensitive to the external environment, namely, the air or water on the nasal epithelium, depending on lifestyle of the animal.

Selective injection system into hippocampus CA1 via monitored theta oscillation.

Methods of cell biology and electrophysiology using dissociated primary cultured neurons allow in vitro study of molecular functions; however, analysis of intact neuronal circuitry is often preferable. To investigate exogenous genes, viral vectors are most commonly injected using a pipette that is inserted from the top of the cortex. Although there are few reports that describe the success rate of injection in detail, it is sometimes difficult to locate the pipette tip accurately within the CA1 pyramidal cell layer because the pyramidal layer is only 0.1 mm thick. In the present study, we have developed a system to inject viral vectors accurately into the mouse hippocampal CA1 pyramidal cell layer using a stereotaxic injection system with simultaneous electrophysiological monitoring of theta oscillation. The pipette tip was positioned reliably based on integrated values of the theta oscillation in the hippocampal CA1 pyramidal cell layer. This approach allows accurate injection of solutions and provides an efficient method of gene transfer using viral vectors into the hippocampus, which can be a useful tool for studies involving the molecular mechanisms of neuronal functions.