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.

Distinct Cell Surface Expression Patterns of N-Glycosylation Site Mutants of AMPA-Type Glutamate Receptor under the Homo-Oligomeric Expression Conditions.

The AMPA-type glutamate receptor (AMPAR) is a homotetrameric or heterotetrameric ion channel composed of various combinations of four subunits (GluA1-4), and its abundance in the synapse determines the strength of synaptic activity. The formation of oligomers in the endoplasmatic reticulum (ER) is crucial for AMPAR subunits' ER-exit and translocation to the cell membrane. Although N-glycosylation on different AMPAR subunits has been shown to regulate the ER-exit of hetero-oligomers, its role in the ER-exit of homo-oligomers remains unclear. In this study, we investigated the role of N-glycans at GluA1N63/N363 and GluA2N370 in ER-exit under the homo-oligomeric expression conditions, whose mutants are known to show low cell surface expressions. In contrast to the N-glycosylation site mutant GluA1N63Q, the cell surface expression levels of GluA1N363Q and GluA2N370Q increased in a time-dependent manner. Unlike wild-type (WT) GluA1, GluA2WT rescued surface GluA2N370Q expression. Additionally, the expression of GluA1N63Q reduced the cell surface expression level of GluA1WT. In conclusion, our findings suggest that these N-glycans have distinct roles in the ER-exit of GluA1 and GluA2 homo-oligomers; N-glycan at GluA1N63 is a prerequisite for GluA1 ER-exit, whereas N-glycans at GluA1N363 and GluA2N370 control the ER-exit rate.

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.

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/.

Autophagy-dependent rhodopsin degradation prevents retinal degeneration in Drosophila.

Recent studies have demonstrated protective roles for autophagy in various neurodegenerative disorders, including the polyglutamine diseases; however, the role of autophagy in retinal degeneration has remained unclear. Accumulation of activated rhodopsin in some Drosophila mutants leads to retinal degeneration, and although it is known that activated rhodopsin is degraded in endosomal pathways in normal photoreceptor cells, the contribution of autophagy to rhodopsin regulation has remained elusive. This study reveals that activated rhodopsin is degraded by autophagy in collaboration with endosomal pathways to prevent retinal degeneration. Light-dependent retinal degeneration in the Drosophila visual system is caused by the knockdown or mutation of autophagy-essential components, such as autophagy-related protein 7 and 8 (atg-7/atg-8), or genes essential for PE (phosphatidylethanolamine) biogenesis and autophagosome formation, including Phosphatidylserine decarboxylase (Psd) and CDP-ethanolamine:diacylglycerol ethanolaminephosphotransferase (Ept). The knockdown of atg-7/8 or Psd/Ept produced an increase in the amount of rhodopsin localized to Rab7-positive late endosomes. This rhodopsin accumulation, followed by retinal degeneration, was suppressed by overexpression of Rab7, which accelerated the endosomal degradation pathway. These results indicate a degree of cross talk between the autophagic and endosomal/lysosomal pathways. Importantly, a reduction in rhodopsin levels rescued Psd knockdown-induced retinal degeneration. Additionally, the Psd knockdown-induced retinal degeneration phenotype was enhanced by Ppt1 inactivation, which causes infantile neuronal ceroid lipofuscinosis, implying that autophagy plays a significant role in its pathogenesis. Collectively, the current data reveal that autophagy suppresses light-dependent retinal degeneration in collaboration with the endosomal degradation pathway and that rhodopsin is a key substrate for autophagic degradation in this context.

KIF4 motor regulates activity-dependent neuronal survival by suppressing PARP-1 enzymatic activity.

In brain development, apoptosis is a physiological process that controls the final numbers of neurons. Here, we report that the activity-dependent prevention of apoptosis in juvenile neurons is regulated by kinesin superfamily protein 4 (KIF4), a microtubule-based molecular motor. The C-terminal domain of KIF4 is a module that suppresses the activity of poly (ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme known to maintain cell homeostasis by repairing DNA and serving as a transcriptional regulator. When neurons are stimulated by membrane depolarization, calcium signaling mediated by CaMKII induces dissociation of KIF4 from PARP-1, resulting in upregulation of PARP-1 activity, which supports neuron survival. After dissociation from PARP-1, KIF4 enters into the cytoplasm from the nucleus and moves to the distal part of neurites in a microtubule-dependent manner. We suggested that KIF4 controls the activity-dependent survival of postmitotic neurons by regulating PARP-1 activity in brain development.