The multifaceted effects of YTHDC1-mediated nuclear m6A recognition.

N6-methyladenosine or m6A modification to mRNAs is now recognised as a key regulator of gene expression and protein translation. The fate of m6A-modified mRNAs is decoded by m6A readers, mostly found in the cytoplasm, except for the nuclear-localised YTHDC1. While earlier studies have implicated YTHDC1-m6A functions in alternative splicing and mRNA export, recent literature has expanded its close association to the chromatin-associated, noncoding and regulatory RNAs to fine-tune transcription and gene expression in cells. Here, we summarise current progress in the study of YTHDC1 function in cells, highlighting its multiple modes of action in regulating gene expression, and propose the formation of YTHDC1 nuclear condensates as a general mechanism that underlies its diverse functions in the nucleus.

Global quantitative proteomic analysis of aged mouse hippocampus.

Understanding the molecular changes associated with the aged brain forms the basis for developing potential strategies for slowing cognitive decline associated with normal aging. Focusing on the hippocampus, a critical brain region involved in learning and memory, we employed tandem mass tag methodology to investigate global proteomic changes that occur in advanced-aged (20-month) versus young (3-month) C57BL/6 male mice. Our analysis revealed the upregulation of 236 proteins in the old hippocampal proteome, including those enriched within several age-related processes, such as the adaptive immune response and molecular metabolic pathways, whereas downregulated proteins (88 in total) are mainly involved in axonogenesis and growth cone-related processes. Categorizing proteins by cell-type enrichment in the brain identified a general upregulation of proteins preferentially expressed in microglia, astrocytes, and oligodendrocytes. In contrast, proteins with neuron-specific expression displayed an overall age-related downregulation. By integrating our proteomic with our previously published transcriptomic data, we discovered a mild but significant positive correlation between mRNA and protein expression changes in the aged hippocampus. Therefore, this proteomic data is a valuable additional resource for further understanding age-related molecular mechanisms.

Ubiquitination of the GluA1 Subunit of AMPA Receptors Is Required for Synaptic Plasticity, Memory, and Cognitive Flexibility.

Activity-dependent changes in the number of AMPA-type glutamate receptors (AMPARs) at the synapse underpin the expression of LTP and LTD, cellular correlates of learning and memory. Post-translational ubiquitination has emerged as a key regulator of the trafficking and surface expression of AMPARs, with ubiquitination of the GluA1 subunit at Lys-868 controlling the post-endocytic sorting of the receptors into the late endosome for degradation, thereby regulating their stability at synapses. However, the physiological significance of GluA1 ubiquitination remains unknown. In this study, we generated mice with a knock-in mutation in the major GluA1 ubiquitination site (K868R) to investigate the role of GluA1 ubiquitination in synaptic plasticity, learning, and memory. Our results reveal that these male mice have normal basal synaptic transmission but exhibit enhanced LTP and deficits in LTD. They also display deficits in short-term spatial memory and cognitive flexibility. These findings underscore the critical roles of GluA1 ubiquitination in bidirectional synaptic plasticity and cognition in male mice.SIGNIFICANCE STATEMENT Subcellular targeting and membrane trafficking determine the precise number of AMPA-type glutamate receptors at synapses, processes that are essential for synaptic plasticity, learning, and memory. Post-translational ubiquitination of the GluA1 subunit marks AMPARs for degradation, but its functional role in vivo remains unknown. Here we demonstrate that the GluA1 ubiquitin-deficient mice exhibit an altered threshold for synaptic plasticity accompanied by deficits in short-term memory and cognitive flexibility. Our findings suggest that activity-dependent ubiquitination of GluA1 fine-tunes the optimal number of synaptic AMPARs required for bidirectional synaptic plasticity and cognition in male mice. Given that increases in amyloid-ß cause excessive ubiquitination of GluA1, inhibiting that GluA1 ubiquitination may have the potential to ameliorate amyloid-ß-induced synaptic depression in Alzheimer's disease.

The m6A-epitranscriptome in brain plasticity, learning and memory.

Activity-dependent gene expression and protein translation underlie the ability of neurons to dynamically adjust their synaptic strength in response to sensory experience and during learning. The emerging field of epitranscriptomics (RNA modifications) has rapidly shifted our views on the mechanisms that regulate gene expression. Among hundreds of biochemical modifications on RNA, N6-methyladenosine (m6A) is the most abundant reversible mRNA modification in the brain. Its dynamic nature and ability to regulate all aspects of mRNA processing have positioned m6A as an important and versatile regulator of nervous system functions, including neuronal plasticity, learning and memory. In this review, we summarise recent experimental evidence that supports the role of m6A signalling in learning and memory, as well as providing an overview of the underlying molecular mechanisms in neurons. We also discuss the consequences of perturbed m6A signalling and/or its regulatory networks which are increasingly being linked to various cognitive disorders in humans.

Identification of phosphorylated tau protein interactors in progressive supranuclear palsy (PSP) reveals networks involved in protein degradation, stress response, cytoskeletal dynamics, metabolic processes, and neurotransmission.

Progressive supranuclear palsy (PSP) is a late-onset neurodegenerative disease defined pathologically by the presence of insoluble phosphorylated-Tau (p-Tau) in neurons and glia. Identifying co-aggregating proteins within p-Tau inclusions may reveal important insights into processes affected by the aggregation of Tau. We used a proteomic approach, which combines antibody-mediated biotinylation and mass spectrometry (MS) to identify proteins proximal to p-Tau in PSP. Using this proof-of-concept workflow for identifying interacting proteins of interest, we characterized proteins proximal to p-Tau in PSP cases, identifying >84% of previously identified interaction partners of Tau and known modifiers of Tau aggregation, while 19 novel proteins not previously found associated with Tau were identified. Furthermore, our data also identified confidently assigned phosphorylation sites that have been previously reported on p-Tau. Additionally, using ingenuity pathway analysis (IPA) and human RNA-seq datasets, we identified proteins previously associated with neurological disorders and pathways involved in protein degradation, stress responses, cytoskeletal dynamics, metabolism, and neurotransmission. Together, our study demonstrates the utility of biotinylation by antibody recognition (BAR) approach to answer a fundamental question to rapidly identify proteins in proximity to p-Tau from post-mortem tissue. The application of this workflow opens up the opportunity to identify novel protein targets to give us insight into the biological process at the onset and progression of tauopathies.

Subunit-Specific Augmentation of AMPA Receptor Ubiquitination by Phorbol Ester.

Excitatory neurotransmission relies on the precise targeting of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors to the neuronal plasma membrane. Activity-dependent ubiquitination of AMPA receptor (AMPAR) subunits sorts internalised receptors to late endosomes for degradation, which ultimately determines the number of AMPARs on neuronal membrane. Our recent study has demonstrated a functional cross-talk between the phosphorylation and ubiquitination of the GluA1 subunit in mammalian central neurons. However, the existence of such a cross modulation for the GluA2 subunit remains unknown. Here, we have shown that bicuculline induced GluA2 ubiquitination on the same lysine residues (Lys-870 and Lys-882) in the C-terminal as those elicited by the AMPA treatment. Interestingly, bicuculline-induced ubiquitination was markedly enhanced by the phospho-mimetic GluA2 S880E mutant. Pharmacological activation of protein kinase C (PKC) by phorbol ester, which mediates the phosphorylation of GluA2 at Ser-880, augmented bicuculline-induced ubiquitination of GluA2 in cultured neurons. This effect was specific for the GluA2 subunit because phorbol ester did not alter the level of GluA1 ubiquitination. However, phorbol ester-induced enhancement of GluA2 ubiquitination did not require Ser-880 phosphorylation. This suggests that pseudo-phosphorylation of Ser-880 is sufficient but is not necessary for the augmentation of bicuculline-induced GluA2 ubiquitination. Collectively, these data provide the first demonstration of subunit-specific modulation of AMPAR ubiquitination by the PKC-dependent signalling pathway in mammalian central neurons.

NFIX-Mediated Inhibition of Neuroblast Branching Regulates Migration Within the Adult Mouse Ventricular-Subventricular Zone.

Understanding the migration of newborn neurons within the brain presents a major challenge in contemporary biology. Neuronal migration is widespread within the developing brain but is also important within the adult brain. For instance, stem cells within the ventricular-subventricular zone (V-SVZ) and the subgranular zone of dentate gyrus of the adult rodent brain produce neuroblasts that migrate to the olfactory bulb and granule cell layer of the dentate gyrus, respectively, where they regulate key brain functions including innate olfactory responses, learning, and memory. Critically, our understanding of the factors mediating neuroblast migration remains limited. The transcription factor nuclear factor I X (NFIX) has previously been implicated in embryonic cortical development. Here, we employed conditional ablation of Nfix from the adult mouse brain and demonstrated that the removal of this gene from either neural stem and progenitor cells, or neuroblasts, within the V-SVZ culminated in neuroblast migration defects. Mechanistically, we identified aberrant neuroblast branching, due in part to increased expression of the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), as a factor contributing to abnormal migration in Nfix-deficient adult mice. Collectively, these data provide new insights into how neuroblast migration is regulated at a transcriptional level within the adult brain.

MicroRNA-mediated disruption of dendritogenesis during a critical period of development influences cognitive capacity later in life.

The prenatal period of cortical development is important for the establishment of neural circuitry and functional connectivity of the brain; however, the molecular mechanisms underlying this process remain unclear. Here we report that disruption of the actin-cytoskeletal network in the developing mouse prefrontal cortex alters dendritic morphogenesis and synapse formation, leading to enhanced formation of fear-related memory in adulthood. These effects are mediated by a brain-enriched microRNA, miR-9, through its negative regulation of diaphanous homologous protein 1 (Diap1), a key organizer of the actin cytoskeletal assembly. Our findings not only revealed important regulation of dendritogenesis and synaptogenesis during early brain development but also demonstrated a tight link between these early developmental events and cognitive functions later in life.

GluA1 subunit ubiquitination mediates amyloid-ß-induced loss of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors.

The accumulation of soluble amyloid-ß (Aß) peptides produces profound neuronal changes in the brain during the pathogenesis of Alzheimer's disease. Excessive levels of Aß disrupt excitatory synaptic transmission by promoting the removal of synaptic AMPA receptors (AMPARs), dendritic spine loss, and synaptic depression. Recently, activity-dependent ubiquitination of the GluA1 subunit has been shown to regulate the intracellular sorting of AMPARs toward late endosomes for degradation. However, whether this ubiquitin signaling pathway mediates Aß-induced loss of surface AMPARs is unknown. In this study, we demonstrate that acute exposure of cultured neurons to soluble Aß oligomers induces AMPAR ubiquitination concomitant with the removal of AMPARs from the plasma membrane. Importantly, expression of the GluA1 ubiquitin-deficient mutants inhibited the adverse effects of Aß on the surface expression of AMPARs in neurons. Furthermore, we revealed the cross-talk between GluA1 ubiquitination and phosphorylation, in particular phosphorylation at Ser-845, which is crucial for AMPAR recycling and is known to be dephosphorylated in the presence of Aß. Our data showed that the GluA1 ubiquitin-deficient mutant enhances GluA1 phosphorylation on Ser-845. Conversely, the GluA1 S845D phosphomimetic mutant reduced binding with Nedd4-1 and hence the ubiquitination of AMPARs. Importantly, the GluA1 S845D mutant also prevented Aß-induced removal of surface AMPARs. Taken together, these findings provide the first demonstration of the dynamic cross-modulation of GluA1 ubiquitination and phosphorylation, a process that is perturbed by Aß, in regulating the membrane sorting decision that ultimately determines the number of AMPARs on the cell surface.

The m6A-epitranscriptomic signature in neurobiology: from neurodevelopment to brain plasticity.

Research over the past decade has provided strong support for the importance of various epigenetic mechanisms, including DNA and histone modifications in regulating activity-dependent gene expression in the mammalian central nervous system. More recently, the emerging field of epitranscriptomics revealed an equally important role of post-transcriptional RNA modifications in shaping the transcriptomic landscape of the brain. This review will focus on the methylation of the adenosine base at the N6 position, termed N6 methyladenosine (m6A), which is the most abundant internal modification that decorates eukaryotic messenger RNAs. Given its prevalence and dynamic regulation in the adult brain, the m6A-epitranscriptome provides an additional layer of regulation on RNA that can be controlled in a context- and stimulus-dependent manner. Conceptually, m6A serves as a molecular switch that regulates various aspects of RNA function, including splicing, stability, localization, or translational control. The versatility of m6A function is typically determined through interaction or disengagement with specific classes of m6A-interacting proteins. Here we review recent advances in the field and provide insights into the roles of m6A in regulating brain function, from development to synaptic plasticity, learning, and memory. We also discuss how aberrant m6A signaling may contribute to neurodevelopmental and neuropsychiatric disorders.

Experience-Dependent Accumulation of N6-Methyladenosine in the Prefrontal Cortex Is Associated with Memory Processes in Mice.

UNLABELLED: The RNA modification N(6)-methyladenosine (m(6)A) influences mRNA stability and cell-type-specific developmental programming, and is highly abundant in the adult brain. However, it has not been determined whether m(6)A is dynamically regulated by experience. Based on transcriptome-wide profiling of m(6)A, we report that the level of m(6)A increases in the medial prefrontal cortex (mPFC) of mice in response to behavioral experience. The modulation was enriched near the stop codon of mRNAs, including genes related to neuronal plasticity. In primary cortical neurons, in vitro, modulation of m(6)A by the RNA demethylase FTO influenced the degradation profiles of a subset of transcripts with modulated sites. In vivo, the expression of Fto and the m(6)A methyltransferase, Mettl3 correlated with the observed increase in m(6)A levels post-training. Furthermore, targeted knockdown of FTO in the mPFC led to enhanced consolidation of cued fear memory. Thus, together with its role in early development, the dynamic regulation of m(6)A in the adult brain serves as an important epitranscriptomic mechanism associated with behavioral adaptation. SIGNIFICANCE STATEMENT: N(6)-methyladenosine (m(6)A) is the most prevalent internal modification on RNA, however, its cellular dynamics in vivo remains elusive. Here we provide the first demonstration of m(6)A upregulation in the mouse medial prefrontal cortex (mPFC) following behavioral training. Knocking down the m(6)A demethylase FTO in the mPFC, which increases total m(6)A level, results in enhanced consolidation of fear memory. Our findings suggest that m(6)A is regulated in an activity-dependent manner in the adult brain, and may function to fine-tune mRNA turnover during memory-related processes.

Neocortical Tet3-mediated accumulation of 5-hydroxymethylcytosine promotes rapid behavioral adaptation.

5-hydroxymethylcytosine (5-hmC) is a novel DNA modification that is highly enriched in the adult brain and dynamically regulated by neural activity. 5-hmC accumulates across the lifespan; however, the functional relevance of this change in 5-hmC and whether it is necessary for behavioral adaptation have not been fully elucidated. Moreover, although the ten-eleven translocation (Tet) family of enzymes is known to be essential for converting methylated DNA to 5-hmC, the role of individual Tet proteins in the adult cortex remains unclear. Using 5-hmC capture together with high-throughput DNA sequencing on individual mice, we show that fear extinction, an important form of reversal learning, leads to a dramatic genome-wide redistribution of 5-hmC within the infralimbic prefrontal cortex. Moreover, extinction learning-induced Tet3-mediated accumulation of 5-hmC is associated with the establishment of epigenetic states that promote gene expression and rapid behavioral adaptation.

PICK1 interacts with PACSIN to regulate AMPA receptor internalization and cerebellar long-term depression.

The dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses is crucial for synaptic transmission, plasticity, learning, and memory. The protein interacting with C-kinase 1 (PICK1) directly interacts with GluA2/3 subunits of the AMPARs. Although the role of PICK1 in regulating AMPAR trafficking and multiple forms of synaptic plasticity is known, the exact molecular mechanisms underlying this process remain unclear. Here, we report a unique interaction between PICK1 and all three members of the protein kinase C and casein kinase II substrate in neurons (PACSIN) family and show that they form a complex with AMPARs. Our results reveal that knockdown of the neuronal-specific protein, PACSIN1, leads to a significant reduction in AMPAR internalization following the activation of NMDA receptors in hippocampal neurons. The interaction between PICK1 and PACSIN1 is regulated by PACSIN1 phosphorylation within the variable region and is required for AMPAR endocytosis. Similarly, the binding of PICK1 to the ubiquitously expressed PACSIN2 is also regulated by the homologous phosphorylation sites within the PACSIN2-variable region. Genetic deletion of PACSIN2, which is highly expressed in Purkinje cells, eliminates cerebellar long-term depression. This deficit can be fully rescued by overexpressing wild-type PACSIN2, but not by a PACSIN2 phosphomimetic mutant, which does not bind PICK1 efficiently. Taken together, our data demonstrate that the interaction of PICK1 and PACSIN is required for the activity-dependent internalization of AMPARs and for the expression of long-term depression in the cerebellum.

Amyloid-ß-Induced Dysregulation of AMPA Receptor Trafficking.

Evidence from neuropathological, genetic, animal model, and biochemical studies has indicated that the accumulation of amyloid-beta (Aß) is associated with, and probably induces, profound neuronal changes in brain regions critical for memory and cognition in the development of Alzheimer's disease (AD). There is considerable evidence that synapses are particularly vulnerable to AD, establishing synaptic dysfunction as one of the earliest events in pathogenesis, prior to neuronal loss. It is clear that excessive Aß levels can disrupt excitatory synaptic transmission and plasticity, mainly due to dysregulation of the AMPA and NMDA glutamate receptors in the brain. Importantly, AMPA receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. This is essential for synaptic plasticity, a cellular correlate of learning and memory, which are the cognitive functions that are most disrupted in AD. Here we review recent advances in the field and provide insights into the molecular mechanisms that underlie Aß-induced dysfunction of AMPA receptor trafficking. This review focuses primarily on NMDA receptor- and metabotropic glutamate receptor-mediated signaling. In particular, we highlight several mechanisms that underlie synaptic long-term depression as common signaling pathways that are hijacked by the neurotoxic effects of Aß.

The nuclear localization pattern and interaction partners of GTF2IRD1 demonstrate a role in chromatin regulation.

GTF2IRD1 is one of the three members of the GTF2I gene family, clustered on chromosome 7 within a 1.8 Mb region that is prone to duplications and deletions in humans. Hemizygous deletions cause Williams-Beuren syndrome (WBS) and duplications cause WBS duplication syndrome. These copy number variations disturb a variety of developmental systems and neurological functions. Human mapping data and analyses of knockout mice show that GTF2IRD1 and GTF2I underpin the craniofacial abnormalities, mental retardation, visuospatial deficits and hypersociability of WBS. However, the cellular role of the GTF2IRD1 protein is poorly understood due to its very low abundance and a paucity of reagents. Here, for the first time, we show that endogenous GTF2IRD1 has a punctate pattern in the nuclei of cultured human cell lines and neurons. To probe the functional relationships of GTF2IRD1 in an unbiased manner, yeast two-hybrid libraries were screened, isolating 38 novel interaction partners, which were validated in mammalian cell lines. These relationships illustrate GTF2IRD1 function, as the isolated partners are mostly involved in chromatin modification and transcriptional regulation, whilst others indicate an unexpected role in connection with the primary cilium. Mapping of the sites of protein interaction also indicates key features regarding the evolution of the GTF2IRD1 protein. These data provide a visual and molecular basis for GTF2IRD1 nuclear function that will lead to an understanding of its role in brain, behaviour and human disease.

GTF2IRD2 from the Williams-Beuren critical region encodes a mobile-element-derived fusion protein that antagonizes the action of its related family members.

GTF2IRD2 belongs to a family of transcriptional regulators (including TFII-I and GTF2IRD1) that are responsible for many of the key features of Williams-Beuren syndrome (WBS). Sequence evidence suggests that GTF2IRD2 arose in eutherian mammals by duplication and divergence from the gene encoding TFII-I. However, in GTF2IRD2, most of the C-terminal domain has been lost and replaced by the domesticated remnant of an in-frame hAT-transposon mobile element. In this first experimental analysis of function, we show that transgenic expression of each of the three family members in skeletal muscle causes significant fiber type shifts, but the GTF2IRD2 protein causes an extreme shift in the opposite direction to the two other family members. Mating of GTF2IRD1 and GTF2IRD2 mice restores the fiber type balance, indicating an antagonistic relationship between these two paralogs. In cells, GTF2IRD2 localizes to cytoplasmic microtubules and discrete speckles in the nuclear periphery. We show that it can interact directly with TFII-Iß and GTF2IRD1, and upon co-transfection changes the normal distribution of these two proteins into a punctate nuclear pattern typical of GTF2IRD2. These data suggest that GTF2IRD2 has evolved as a regulator of GTF2IRD1 and TFII-I; inhibiting their function by direct interaction and sequestration into inactive nuclear zones.

The N6-methyladenosine RNA landscape in the aged mouse hippocampus.

The aged brain is associated with an inevitable decline in cognitive function and increased vulnerability to neurodegenerative disorders. Multiple molecular hallmarks have been associated with the aging nervous system through transcriptomics and proteomic studies. Recently, epitranscriptomic analysis has highlighted the role of RNA chemical modification in various biological processes. In particular, N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNAs, has been functionally linked to multiple aspects of RNA metabolism with the roles of m6A in processes such as learning and memory, leading to our current investigation of how the m6A-transcriptomic landscape is shaped during aging. Using the inbred C57BL/6 line, we compared the m6A-transcriptomic profiles from the hippocampi of young (3-month-old) and aged (20-month-old) mice. Methylated RNA immunoprecipitation (MeRIP)-sequencing analysis revealed hyper- and hypomethylation in 426 and 102 genes, respectively, in the aged hippocampus (fold change >1.5, false discovery rate <0.05). By correlating the methylation changes to their steady-state transcript levels in the RNA-Seq data, we found a significant concordance between m6A and transcript levels in both directions. Notably, the myelin regulator gene Gpr17 was downregulated in the aged hippocampus concomitant with reduced m6A levels in its 3'UTR. Using reporter constructs and mutagenesis analysis, we demonstrated that the putative m6A sites in the 3'UTR of Gpr17 are important for mRNA translation but not for regulating transcript stability. Overall, the positive correlation between m6A and the transcript expression levels indicates a co-transcriptional regulation of m6A with gene expression changes that occur in the aged mouse hippocampus.

Different Low-complexity Regions of SFPQ Play Distinct Roles in the Formation of Biomolecular Condensates.

Demixing of proteins and nucleic acids into condensed liquid phases is rapidly emerging as a ubiquitous mechanism underlying the complex spatiotemporal organisation of molecules within the cell. Long disordered regions of low sequence complexity (LCRs) are a common feature of proteins that form liquid-like microscopic biomolecular condensates. In particular, RNA-binding proteins with prion-like regions have emerged as key drivers of liquid demixing to form condensates such as nucleoli, paraspeckles and stress granules. Splicing factor proline- and glutamine-rich (SFPQ) is an RNA- and DNA-binding protein essential for DNA repair and paraspeckle formation. SFPQ contains two LCRs of different length and composition. Here, we show that the shorter C-terminal LCR of SFPQ is the main region responsible for the condensation of SFPQ in vitro and in the cell nucleus. In contrast, we find that the longer N-terminal prion-like LCR of SFPQ attenuates condensation of the full-length protein, suggesting a more regulatory role in preventing aberrant condensate formation in the cell. The compositions of these respective LCRs are discussed with reference to current literature. Our data add nuance to the emerging understanding of biomolecular condensation, by providing the first example of a common multifunctional nucleic acid-binding protein with an extensive prion-like region that serves to regulate rather than drive condensate formation.

Platelet-derived exerkine CXCL4/platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice.

The beneficial effects of physical activity on brain ageing are well recognised, with exerkines, factors that are secreted into the circulation in response to exercise, emerging as likely mediators of this response. However, the source and identity of these exerkines remain unclear. Here we provide evidence that an anti-geronic exerkine is secreted by platelets. We show that platelets are activated by exercise and are required for the exercise-induced increase in hippocampal precursor cell proliferation in aged mice. We also demonstrate that increasing the systemic levels of the platelet-derived exerkine CXCL4/platelet factor 4 (PF4) ameliorates age-related regenerative and cognitive impairments in a hippocampal neurogenesis-dependent manner. Together these findings highlight the role of platelets in mediating the rejuvenating effects of exercise during physiological brain ageing.

Copine-6 is a Ca2+ sensor for activity-induced AMPA receptor exocytosis.

The recruitment of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors underlies the strengthening of neuronal connectivity during learning and memory. This process is triggered by N-methyl-D-aspartate (NMDA) receptor-dependent postsynaptic Ca2+ influx. Synaptotagmin (Syt)-1 and -7 have been proposed as Ca2+ sensors for AMPA receptor exocytosis but are functionally redundant. Here, we identify a cytosolic C2 domain-containing Ca2+-binding protein, Copine-6, that forms a complex with AMPA receptors. Loss of Copine-6 expression impairs activity-induced exocytosis of AMPA receptors in primary neurons, which is rescued by wild-type Copine-6 but not Ca2+-binding mutants. In contrast, Copine-6 loss of function does not affect steady-state expression or tetrodotoxin-induced synaptic upscaling of surface AMPA receptors. Loss of Syt-1/Syt-7 significantly reduces Copine-6 protein expression. Interestingly, overexpression of wild-type Copine-6, but not the Ca2+-binding mutants, restores activity-dependent exocytosis of AMPA receptors in Syt-1/Syt-7 double-knockdown neurons. We conclude that Copine-6 is a postsynaptic Ca2+ sensor that mediates AMPA receptor exocytosis during synaptic potentiation.