AMPA Receptor Recycling Research Articles

Neonatal Hypertonia and Progressive Respiratory Failure due to Novel Heterozygous Mutation in ATAD1

ATAD1-related encephalopathy is a novel monogenic disorder first reported in 2017. 1 Ahrens-Nicklas Rebecca C. Umanah George K.E. Neal Sondheimer Deardorff Matthew A. Wilkens Alisha B. Conlin Laura K. Santani Avni B. et al. “Precision Therapy for a New Disorder of AMPA Receptor Recycling Due to Mutations in ATAD1.”. Neurology Genetics. February 1, 2017; 3https://doi.org/10.1212/NXG.0000000000000130 Crossref Scopus (30) Google Scholar ATAD1 encodes a thorase involved in mitochondrial and peroxisome function that regulates post-synaptic alpha-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptors (AMPAR). 2 Piard Juliette Essien Umanah George K Harms Frederike L Abalde-Atristain Leire Daniel Amram Chang Melissa Chen Rong et al. “A Homozygous ATAD1 Mutation Impairs Postsynaptic AMPA Receptor Trafficking and Causes a Lethal Encephalopathy.”. Brain. March 1, 2018; 141 (61): 651https://doi.org/10.1093/brain/awx377 Crossref Scopus (52) Google Scholar There are eight confirmed cases of ATAD1-related encephalopathy in five families due to three loss of function and one gain of function mutations. 2 Piard Juliette Essien Umanah George K Harms Frederike L Abalde-Atristain Leire Daniel Amram Chang Melissa Chen Rong et al. “A Homozygous ATAD1 Mutation Impairs Postsynaptic AMPA Receptor Trafficking and Causes a Lethal Encephalopathy.”. Brain. March 1, 2018; 141 (61): 651https://doi.org/10.1093/brain/awx377 Crossref Scopus (52) Google Scholar ,3 Bunod Roxane Doummar Diane Whalen Sandra Keren Boris Chantot-Bastaraud Sandra Kim Maincent Marie-Charlotte Villy et al. “Congenital Immobility and Stiffness Related to Biallelic ATAD1 Variants.”. Neurology Genetics. December 1, 2020; 6https://doi.org/10.1212/NXG.0000000000000520 Crossref Scopus (1) Google Scholar All but one family were consanguineous and all patients had homozygous mutations. 3 Bunod Roxane Doummar Diane Whalen Sandra Keren Boris Chantot-Bastaraud Sandra Kim Maincent Marie-Charlotte Villy et al. “Congenital Immobility and Stiffness Related to Biallelic ATAD1 Variants.”. Neurology Genetics. December 1, 2020; 6https://doi.org/10.1212/NXG.0000000000000520 Crossref Scopus (1) Google Scholar The syndrome is characterized by progressive peripheral hypertonia with onset at birth, absent or near absent spontaneous movements, respiratory distress and other symptoms including tremor, clinical seizures, feeding difficulties, hernias, abnormal electroencephalogram (EEG), normal initial neuroimaging with varied abnormalities at later age, and abnormal brainstem auditory evoked response test. 1 Ahrens-Nicklas Rebecca C. Umanah George K.E. Neal Sondheimer Deardorff Matthew A. Wilkens Alisha B. Conlin Laura K. Santani Avni B. et al. “Precision Therapy for a New Disorder of AMPA Receptor Recycling Due to Mutations in ATAD1.”. Neurology Genetics. February 1, 2017; 3https://doi.org/10.1212/NXG.0000000000000130 Crossref Scopus (30) Google Scholar , 2 Piard Juliette Essien Umanah George K Harms Frederike L Abalde-Atristain Leire Daniel Amram Chang Melissa Chen Rong et al. “A Homozygous ATAD1 Mutation Impairs Postsynaptic AMPA Receptor Trafficking and Causes a Lethal Encephalopathy.”. Brain. March 1, 2018; 141 (61): 651https://doi.org/10.1093/brain/awx377 Crossref Scopus (52) Google Scholar , 3 Bunod Roxane Doummar Diane Whalen Sandra Keren Boris Chantot-Bastaraud Sandra Kim Maincent Marie-Charlotte Villy et al. “Congenital Immobility and Stiffness Related to Biallelic ATAD1 Variants.”. Neurology Genetics. December 1, 2020; 6https://doi.org/10.1212/NXG.0000000000000520 Crossref Scopus (1) Google Scholar , 4 Wolf Nicole I Zschocke Johannes Jakobs Cornelis Dietz Rating Hoffmann Georg F “ATAD1 Encephalopathy and Stiff Baby Syndrome: A Recognizable Clinical Presentation.”. Brain. June 1, 2018; 141: e49https://doi.org/10.1093/brain/awy095 Crossref Scopus (4) Google Scholar Loss of ATAD1 disrupts internalization of post-synaptic AMPA receptors, which result in excessive glutamatergic signaling in the brain. 2 Piard Juliette Essien Umanah George K Harms Frederike L Abalde-Atristain Leire Daniel Amram Chang Melissa Chen Rong et al. “A Homozygous ATAD1 Mutation Impairs Postsynaptic AMPA Receptor Trafficking and Causes a Lethal Encephalopathy.”. Brain. March 1, 2018; 141 (61): 651https://doi.org/10.1093/brain/awx377 Crossref Scopus (52) Google Scholar In the original report, Perampanel, an AMPAR antagonist, was trialed based on successful mouse data with improvement in patients’ hypertonicity and seizure resolution. 1 Ahrens-Nicklas Rebecca C. Umanah George K.E. Neal Sondheimer Deardorff Matthew A. Wilkens Alisha B. Conlin Laura K. Santani Avni B. et al. “Precision Therapy for a New Disorder of AMPA Receptor Recycling Due to Mutations in ATAD1.”. Neurology Genetics. February 1, 2017; 3https://doi.org/10.1212/NXG.0000000000000130 Crossref Scopus (30) Google Scholar Among prior cases, six of eight patients died of respiratory failure and/or cardiac sequalae of respiratory distress at a mean age of five months. 3 Bunod Roxane Doummar Diane Whalen Sandra Keren Boris Chantot-Bastaraud Sandra Kim Maincent Marie-Charlotte Villy et al. “Congenital Immobility and Stiffness Related to Biallelic ATAD1 Variants.”. Neurology Genetics. December 1, 2020; 6https://doi.org/10.1212/NXG.0000000000000520 Crossref Scopus (1) Google Scholar This case highlights unsuccessful Perampanel use in an infant with ATAD1-related encephalopathy.

Characterizing a Novel RGS14:MyosinV/VI Interaction in Brain

RGS14 is a complex multifunctional signaling protein that integrates G protein, Ras/ERK and Ca++/CaM/CaMKII signaling pathways. The protein is composed of an RGS domain that binds active Gαi/o, two tandem Ras/Rap binding domains (R1, R2) which bind active H‐Ras‐GTP and Rap2A‐GTP to regulate MAP kinase signaling, a G protein regulatory (GPR) motif that binds inactive Gαi1/3, and a Ca++/CaM binding site. RGS14 is highly expressed in the hippocampus, the brain center for learning and memory, which is composed of areas CA3, CA2, and CA1. Schaffer collateral (SC) projections from CA3 to CA1 display long term potentiation (LTP), a stable potentiation in post‐synaptic strength in response to brief periods of synaptic stimulation such as during learning in animals. Within the hippocampus RGS14 is highly expressed in postsynaptic dendrites and spines of area CA2, where SC inputs from CA3 to CA2 neurons show a surprising resistance to LTP. CA2 has been shown to be important for social learning and memory, and we have shown that RGS14 is a natural suppressor of LTP and synaptic plasticity in this area. That is, loss of RGS14 in CA2 (RGS14‐KO) restores synaptic plasticity (LTP) in CA2 and markedly enhances hippocampal learning. However, it remains unknown how RGS14 interacts with other signaling proteins to regulate synaptic plasticity. In proteomic studies, RGS14 recovered from mouse brain by immunoprecipitation was subjected to mass spectrometry and proteomic analysis, showing its association with a high molecular‐weight protein complex including actin‐based myosin motor proteins (Myosin V, Myosin VI and Myosin II) and their regulatory proteins, among others. MyoV is involved in activity‐dependent insertion of AMPA glutamate receptor subunits (GluR1, GluR2) and NMDA receptors, regulation of calcium and proper positioning of calcium intracellular stores. MyoVI is enriched at the postsynaptic density where it regulates AMPA receptor recycling. These myosin classes have crucial roles in regulating synaptic plasticity and LTP. Follow up studies have shown RGS14 in complex with MyoV and MyoVI in whole brain lysates. However, it remains unclear how RGS14 interacts with MyoV/VI to regulate synaptic plasticity. Ongoing studies will determine if RGS14 binds directly or indirectly to MyoV/VI and how that is regulated, as well as elucidate the physiological function of this interaction, thereby providing insight into the functions of both RGS14 and MyoV/VI in their roles in modulating synaptic plasticity and hippocampal‐dependent learning and memory.Support or Funding InformationThis research was supported by grants NIH/NINDS to JRH (5R01NS37112 and 1R21NS074975), and in part by the Emory Neuroscience NINDS Core Facilities and NIH/NINDS under award number P30NS055077.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Disease-Associated Chaperone FKBP51 Impairs Cognitive Function by Accelerating AMPA Receptor Recycling.

Increased expression of the FK506-binding protein 5 (FKBP5) gene has been associated with a number of diseases, but most prominently in connection to psychiatric illnesses. Many of these psychiatric disorders present with dementia and other cognitive deficits, but a direct connection between these issues and alterations in FKBP5 remains unclear. We generated a novel transgenic mouse to selectively overexpress FKBP5, which encodes the FKBP51 protein, in the corticolimbic system, which had no overt effects on gross body weight, motor ability, or general anxiety. Instead, we found that overexpression of FKBP51 impaired long-term depression (LTD) as well as spatial reversal learning and memory, suggesting a role in glutamate receptor regulation. Indeed, FKBP51 altered the association of heat-shock protein 90 (Hsp90) with AMPA receptors, which was accompanied by an accelerated rate of AMPA recycling. In this way, the chaperone system is critical in triage decisions for AMPA receptor trafficking. Imbalance in the chaperone system may manifest in impairments in both inhibitory learning and cognitive function. These findings uncover an unexpected and essential mechanism for learning and memory that is controlled by the psychiatric risk factor FKBP5.

Stargazin Dephosphorylation Mediates Homeostatic Synaptic Downscaling of Excitatory Synapses.

Synaptic scaling is a form of homeostatic plasticity that is critical for maintaining neuronal activity within a dynamic range, and which alters synaptic strength through changes in postsynaptic AMPA-type glutamate receptors. Homeostatic scaling down of excitatory synapses has been shown to occur during sleep, and to contribute to synapse remodeling and memory consolidation, but the underlying mechanisms are only partially known. Here, we report that synaptic downscaling in cortical neurons is accompanied by dephosphorylation of the transmembrane AMPA receptor regulatory protein stargazin, and by an increase in its cell surface mobility. The changes in stargazin surface diffusion were paralleled by an increase in the mobility of GluA1-containing AMPA receptors at synaptic sites. In addition, stargazin dephosphorylation was required for the downregulation of surface levels of GluA1-containing AMPA receptors promoted by chronic elevation of neuronal activity, specifically by mediating the interaction with the adaptor proteins AP-2 and AP-3A. Disruption of the stargazin-AP-3A interaction was sufficient to prevent the decrease in cell surface GluA1-AMPA receptor levels associated with chronically enhanced synaptic activity, suggesting that scaling down is accomplished through decreased AMPA receptor recycling and enhanced lysosomal degradation. Thus, synaptic downscaling is associated with both increased stargazin and AMPA receptor cell surface diffusion, as well as with stargazin-mediated AMPA receptor endocytosis and lysosomal degradation.

Recent Findings on AMPA Receptor Recycling.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) are tetrameric protein complexes that mediate most of the fast-excitatory transmission in response to the neurotransmitter glutamate in neurons. The abundance of AMPA-Rs at the surface of excitatory synapses establishes the strength of the response to glutamate. It is thus evident that neurons need to tightly regulate this feature, particularly in the context of all synaptic plasticity events, which are considered the biological correlates of higher cognitive functions such as learning and memory. AMPA-R levels at the synapse are regulated by insertion of newly synthesized receptors, lateral diffusion on the plasma membrane and endosomal cycling. The latter is likely the most important especially for synaptic plasticity. This process starts with the endocytosis of the receptor from the cell surface and is followed by either degradation, if the receptor is directed to the lysosomal compartment, or reinsertion at the cell surface through a specialized endosomal compartment called recycling endosomes. Although the basic steps of this process have been discovered, the details and participation of additional regulatory proteins are still being discovered. In this review article, we describe the most recent findings shedding light on this crucial mechanism of synaptic regulation.

Sorting out memory and learning.

Mutations in AMPA receptor recycling machinery underlie cognitive impairment in an intellectual disorder and in neurodegenerative diseases.

GRASP1 Regulates Synaptic Plasticity and Learning through Endosomal Recycling of AMPA Receptors

Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects onAMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity invivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders.

Metabotropic action of postsynaptic kainate receptors triggers hippocampal long-term potentiation.

Long-term potentiation (LTP) in the rat hippocampus is the most extensively studied cellular model for learning and memory. Induction of classical LTP involves an NMDA-receptor- and calcium-dependent increase in functional synaptic AMPA receptors, mediated by enhanced recycling of internalized AMPA receptors back to the postsynaptic membrane. Here we report a physiologically relevant NMDA-receptor-independent mechanism that drives increased AMPA receptor recycling and LTP. This pathway requires the metabotropic action of kainate receptors and activation of G protein, protein kinase C and phospholipase C. Like classical LTP, kainate-receptor-dependent LTP recruits recycling endosomes to spines, enhances synaptic recycling of AMPA receptors to increase their surface expression and elicits structural changes in spines, including increased growth and maturation. These data reveal a new and, to our knowledge, previously unsuspected role for postsynaptic kainate receptors in the induction of functional and structural plasticity in the hippocampus.

PACSIN1 regulates the dynamics of AMPA receptor trafficking.

Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons.

The memory gene KIBRA is a bidirectional regulator of synaptic and structural plasticity in the adult brain

Memory formation is associated with activity-dependent changes in synaptic plasticity. The mechanisms underlying these processes are complex and involve multiple components. Recent work has implicated the protein KIBRA in human memory, but its molecular functions in memory processes remain not fully understood. Here, we show that a selective overexpression of KIBRA in neurons increases hippocampal long-term potentiation (LTP) but prevents the induction of long-term depression (LTD), and impairs spatial long-term memory in adult mice. KIBRA overexpression increases the constitutive recycling of AMPA receptors containing GluA1 (GluA1-AMPARs), and favors their activity-dependent surface expression. It also results in dramatic dendritic rearrangements in pyramidal neurons both in vitro and in vivo. KIBRA knockdown in contrast, abolishes LTP, decreases GluA1-AMPARs recycling and reduces dendritic arborization. These results establish KIBRA as a novel bidirectional regulator of synaptic and structural plasticity in hippocampal neurons, and of long-term memory, highly relevant to cognitive processes and their pathologies.

Regulation of AMPA receptor subunit GluA1 surface expression by PAK3 phosphorylation

AMPA receptors (AMPARs) are the major excitatory receptors of the brain and are fundamental to synaptic plasticity, memory, and cognition. Dynamic recycling of AMPARs in neurons is regulated through several types of posttranslational modification, including phosphorylation. Here, we identify a previously unidentified signal transduction cascade that modulates phosphorylation of serine residue 863 (S863) in the GluA1 AMPAR subunit and controls surface trafficking of GluA1 in neurons. Activation of the EphR-Ephrin signal transduction pathway enhances S863 phosphorylation. Further, EphB2 can interact with Zizimin1, a guanine-nucleotide exchange factor that activates Cdc42 and stimulates S863 phosphorylation in neurons. Among the numerous targets downstream of Cdc42, we determined that the p21-activated kinase-3 (PAK3) phosphorylates S863 in vitro. Moreover, specific loss of PAK3 expression and pharmacological inhibition of PAK both disrupt activity-dependent phosphorylation of S863 in cortical neurons. EphB2, Cdc42, and PAKs are broadly capable of controlling dendritic spine formation and synaptic plasticity and are implicated in multiple cognitive disorders. Collectively, these data delineate a novel signal cascade regulating AMPAR trafficking that may contribute to the molecular mechanisms that govern learning and cognition.

The X-linked mental retardation protein OPHN1 interacts with Homer1b/c to control spine endocytic zone positioning and expression of synaptic potentiation.

At glutamatergic synapses, local endocytic recycling of AMPA receptors (AMPARs) is important for the supply of a mobile pool of AMPARs required for synaptic potentiation. This local recycling of AMPARs critically relies on the presence of an endocytic zone (EZ) near the postsynaptic density (PSD). The precise mechanisms that couple the EZ to the PSD still remain largely elusive, with the large GTPase Dynamin-3 and the multimeric PSD adaptor protein Homer1 as the two main players identified. Here, we demonstrate that a physical interaction between the X-linked mental retardation protein oligophrenin-1 (OPHN1) and Homer1b/c is crucial for the positioning of the EZ adjacent to the PSD, and present evidence that this interaction is important for OPHN1's role in controlling activity-dependent strengthening of excitatory synapses in the rat hippocampus. Disruption of the OPHN1-Homer1b/c interaction causes a displacement of EZs from the PSD, along with impaired AMPAR recycling and reduced AMPAR accumulation at synapses, in both basal conditions and conditions that can induce synaptic potentiation. Together, our findings unveil a novel role for OPHN1 as an interaction partner of Homer1b/c in spine EZ positioning, and provide new mechanistic insight into how genetic deficits in OPHN1 can lead to impaired synapse maturation and plasticity.

Reply to Wilkinson et al.: Concerning the use of pHluorin-tagged GluA2 as a reporter for NMDA-induced AMPA receptor recycling

As pointed out in the letter by Wilkinson et al. (1), we question in our recent paper by Rathje et al. (2) the use of pHluorin-GluA2 (pH-GluA2) as a reporter of NMDA- and AMPA-induced endocytosis because application of the agonists NMDA and AMPA results in intracellular acidification of hippocampal neurons. This conclusion rests on two important key observations: (i) the pHluorin response to NMDA treatment was almost intact after removal of surface-exposed pHluorin by thrombin cleavage (figure 1 of Rathje et al.) (2) and (ii) NMDA stimulation reduces fluorescence of both ER-retained pH-GluA2 and cytosolic pHluorin, in agreement with intracellular acidification, which was further supported by measurements using the pH-sensitive dye SNARF-1 (figures 2 and 3 of Rathje et al.) (2).

Shank3-Rich2 Interaction Regulates AMPA Receptor Recycling and Synaptic Long-Term Potentiation

Synaptic long-term potentiation (LTP) is a key mechanism involved in learning and memory, and its alteration is associated with mental disorders. Shank3 is a major postsynaptic scaffolding protein that orchestrates dendritic spine morphogenesis, and mutations of this protein lead to mental retardation and autism spectrum disorders. In the present study we investigated the role of a new Shank3-associated protein in LTP. We identified the Rho-GAP interacting CIP4 homolog 2 (Rich2) as a new Shank3 partner by proteomic screen. Using single-cell bioluminescence resonance energy transfer microscopy, we found that Rich2-Shank3 interaction is increased in dendritic spines of mouse cultured hippocampal neurons during LTP. We further characterized Rich2 as an endosomal recycling protein that controls AMPA receptor GluA1 subunit exocytosis and spine morphology. Knock-down of Rich2 with siRNA, or disruption of the Rich2-Shank3 complex using an interfering mimetic peptide, inhibited the dendritic spine enlargement and the increase in GluA1 subunit exocytosis typical of LTP. These results identify Rich2-Shank3 as a new postsynaptic protein complex involved in synaptic plasticity.

Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions

The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing stabilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synaptically during postnatal development and thus could mediate the stabilization of end plate AChR.

GRASP-1 regulates endocytic receptor recycling and synaptic plasticity

Remodeling of synapses is a fundamental mechanism for information storage and processing in the brain. Previous studies showed that the endosomal pathway play a central role in synapse formation and plasticity. A popular model holds that recycling endosomes in dendrites provide the local intracellular pool of postsynaptic receptors for long-term potentiation (LTP), a widely studied cellular model for learning and memory formation. However, we are far from a complete understanding how endocytic receptor sorting and recycling is organized and coordinated in dendrites. Especially, the molecular mechanisms that couple specific endosomal trafficking routes during LTP are poorly understood. In a recent paper we discovered that the coiled-coil protein GRIP-associated protein-1 (GRASP-1) is a neuron-specific effector of the small GTPase Rab4 and key component of AMPA receptor recycling machinery in dendrites.1 GRASP-1 is essential for maintenance of spine morphology and important for LTP. GRASP-1 connects Rab4 and Rab11 recycling endosomal domains through the interaction with target (t)-SNARE syntaxin 13, which constitutes a new principle for regulating endosomal recycling. Here, we summarize our recently reported observations and further discuss their possible implications.

Neuron Specific Rab4 Effector GRASP-1 Coordinates Membrane Specialization and Maturation of Recycling Endosomes

Author SummaryNeurons communicate with each other through specialized structures called synapses, and proper synapse function is fundamental for information processing and memory storage. The endosomal membrane trafficking pathway is crucial for the structure and function of synapses; however, the components of the neuronal endosomal transport machinery are poorly characterized. In this paper, we report that a protein called GRASP-1 is required for neurotransmitter receptor recycling through endosomes and back to the cell surface, as well as for the normal morphology of dendritic spines—the projections that form synapses—and for synaptic plasticity. We show that GRASP-1 coordinates coupling between early and later steps of the endocytic recycling pathway by binding to Rab4, a regulator of early endosomes, and to another endosomal protein found later in the pathway called syntaxin 13—a so-called SNARE protein involved in membrane fusion. GRASP-1 binds Rab4 with its N terminus and syntaxin 13 with its C terminus, suggesting that these interactions could structurally and functionally link early endosomes to those later in the recycling pathway. We propose a model in which GRASP-1 forms a molecular bridge between different endosomal membranes and the SNARE fusion machinery. Our study thus provides new mechanistic information about endosome function in neurons and highlights GRASP-1 as a key molecule that controls membrane receptor sorting and recycling during synaptic plasticity.

Gene expression changes in an animal model of in utero irradiation‐induced cortical dysplasia

Cortical Dysplasia (CD) is the histopathological substrate in almost half of all drug-resistant focal epilepsies. Little is known about the gene expression profile of CD. As such information may help target therapeutics more effectively, our aim was to perform a gene expression analysis of an animal model of cortical dysplasia induced by in utero irradiation. Nine offspring from irradiated animals, and nine age-matched controls were sacrificed at post-natal day 60. Cortical and hippocampal regions were separated, and total ribonucleic acid (RNA) was extracted using a commercially available kit (Qiagen). RNA was then subjected to a gene expression analysis using an oligonucleotide microarray platform (Illumina). After statistical analysis, genes were considered differentially expressed when a p value less than 0.001 was observed. Real-time, quantitative polymerase chain reaction (RT-qPCR) was used to confirm microarray results for three genes via the Livak method. Twenty three genes from cortical tissue met criteria for altered gene expression. Six genes from cortex seemed relevant to the pathogenesis of CD. Two genes that promoted cell survival (connective tissue growth factor and peroxiredoxin) were upregulated. One gene that promoted excitotoxic neurodegeneration (latrophilin-2) was downregulated. Two genes involved in glutamate (protein kinase C-alpha) and AMPA receptor recycling (NEEP-21) were downregulated. One gene, (Shank-1) involved in the control of dendritic maturation, was downregulated. Gene expression analysis in this animal model revealed some of the potential mechanisms by which CD may lead to the phenotype of intractable epilepsy. The downregulation of genes that are involved in glutamate and AMPA receptor recycling may lead to increased excitability. Disinhibition of aberrant dendritic branching, resulting from a downregulation of Shank-1, may also result in an increase in sprouting, excitation and/or hypersynchrony. Finally, genes promoting cell survival, either directly (connective tissue growth factor, peroxiredoxin) or indirectly (latrophilin-2) may allow CD tissue to survive the excitotoxic injury that it produces, thus allowing it to perpetuate the epileptic condition over time.

Modeling the role of lateral membrane diffusion in AMPA receptor trafficking along a spiny dendrite

AMPA receptor trafficking in dendritic spines is emerging as a major postsynaptic mechanism for the expression of plasticity at glutamatergic synapses. AMPA receptors within a spine are in a continuous state of flux, being exchanged with local intracellular pools via exo/endocytosis and with the surrounding dendrite via lateral membrane diffusion. This suggests that one cannot treat a single spine in isolation. Here we present a model of AMPA receptor trafficking between multiple dendritic spines distributed along the surface of a dendrite. Receptors undergo lateral diffusion within the dendritic membrane, with each spine acting as a spatially localized trap where receptors can bind to scaffolding proteins or be internalized through endocytosis. Exocytosis of receptors occurs either at the soma or at sites local to dendritic spines via constitutive recycling from intracellular pools. We derive a reaction-diffusion equation for receptor trafficking that takes into account these various processes. Solutions of this equation allow us to calculate the distribution of synaptic receptor numbers across the population of spines, and hence determine how lateral diffusion contributes to the strength of a synapse. A number of specific results follow from our modeling and analysis. (1) Lateral membrane diffusion alone is insufficient as a mechanism for delivering AMPA receptors from the soma to distal dendrites. (2) A source of surface receptors at the soma tends to generate an exponential-like distribution of receptors along the dendrite, which has implications for synaptic democracy. (3) Diffusion mediates a heterosynaptic interaction between spines so that local changes in the constitutive recycling of AMPA receptors induce nonlocal changes in synaptic strength. On the other hand, structural changes in a spine following long term potentiation or depression have a purely local effect on synaptic strength. (4) A global change in the rates of AMPA receptor exo/endocytosis is unlikely to be the sole mechanism for homeostatic synaptic scaling. (5) The dynamics of AMPA receptor trafficking occurs on multiple timescales and varies according to spatial location along the dendrite. Understanding such dynamics is important when interpreting data from inactivation experiments that are used to infer the rate of relaxation to steady-state.

Endocytosis and recycling of AMPA receptors lacking GluR2/3

Excitatory synapses in the mammalian brain contain two types of ligand-gated ion channels: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). AMPARs are responsible for generating excitatory synaptic responses, whereas NMDAR activation triggers long-lasting changes in these responses by modulating the trafficking of AMPARs toward and away from synapses. AMPARs are tetramers composed of four subunits (GluR1-GluR4), which current models suggest govern distinct AMPAR trafficking behavior during synaptic plasticity. Here, we address the roles of GluR2 and GluR3 in controlling the recycling- and activity-dependent endocytosis of AMPARs by using cultured hippocampal neurons prepared from knockout (KO) mice lacking these subunits. We find that synapses and dendritic spines form normally in cells lacking GluR2/3 and that upon NMDAR activation, GluR2/3-lacking AMPARs are endocytosed in a manner indistinguishable from GluR2-containing AMPARs in wild-type (WT) neurons. AMPARs lacking GluR2/3 also recycle to the plasma membrane identically to WT AMPARs. However, because of their permeability to calcium, GluR2-lacking but not WT AMPARs exhibited robust internalization throughout the dendritic tree in response to AMPA application. Dendritic endocytosis of AMPARs also was observed in GABAergic neurons, which express a high proportion of GluR2-lacking AMPARs. These results demonstrate that GluR2 and GluR3 are not required for activity-dependent endocytosis of AMPARs and suggest that the most important property of GluR2 in the context of AMPAR trafficking may be its influence on calcium permeability.