m6A facilitates hippocampus-dependent learning and memory through YTHDF1.

N6-methyladenosine (m6A), the most prevalent internal RNA modification on mammalian messenger RNAs, regulates the fates and functions of modified transcripts through m6A-specific binding proteins1-5. In the nervous system, m6A is abundant and modulates various neural functions6-11. Whereas m6A marks groups of mRNAs for coordinated degradation in various physiological processes12-15, the relevance of m6A for mRNA translation in vivo remains largely unknown. Here we show that, through its binding protein YTHDF1, m6A promotes protein translation of target transcripts in response to neuronal stimuli in the adult mouse hippocampus, thereby facilitating learning and memory. Mice with genetic deletion of Ythdf1 show learning and memory defects as well as impaired hippocampal synaptic transmission and long-term potentiation. Re-expression of YTHDF1 in the hippocampus of adult Ythdf1-knockout mice rescues the behavioural and synaptic defects, whereas hippocampus-specific acute knockdown of Ythdf1 or Mettl3, which encodes the catalytic component of the m6A methyltransferase complex, recapitulates the hippocampal deficiency. Transcriptome-wide mapping of YTHDF1-binding sites and m6A sites on hippocampal mRNAs identified key neuronal genes. Nascent protein labelling and tether reporter assays in hippocampal neurons showed that YTHDF1 enhances protein synthesis in a neuronal-stimulus-dependent manner. In summary, YTHDF1 facilitates translation of m6A-methylated neuronal mRNAs in response to neuronal stimulation, and this process contributes to learning and memory.

Lack of support for surface diffusion of postsynaptic AMPARs in tuning synaptic transmission.

Repetitive stimulation of excitatory synapses triggers molecular events required for signal transfer across neuronal synapses. It has been hypothesized that one of these molecular events, the diffusion of extrasynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) (i.e., the diffusion hypothesis), is necessary to help synapses recover from paired-pulse depression. To examine this presumed role of AMPAR diffusion during repetitive presynaptic stimulation, a biophysical model based on published physiological results was developed to track the localization and gating of each AMPAR. The model demonstrates that AMPAR gating in short intervals of fewer than 100 ms is controlled by their position in relation to the glutamate release site and by their recovery from desensitization, but it is negligibly influenced by their diffusion. Therefore, these simulations failed to demonstrate a role for AMPAR diffusion in helping synapses recover from paired-pulse depression.

Rapid homeostatic downregulation of LTP by extrasynaptic GluN2B receptors.

Although the activation of extrasynaptic GluN2B-containing N-methyl-d-aspartate (NMDA) receptors has been implicated in neurodegenerative diseases, such as Alzheimer's and Huntington's disease, their physiological function remains unknown. In this study, we found that extrasynaptic GluN2B receptors play a homeostatic role by antagonizing long-term potentiation (LTP) induction under conditions of prolonged synaptic stimulation. In particular, we have previously found that brief theta-pulse stimulation (5 Hz for 30 s) triggers robust LTP, whereas longer stimulation times (5 Hz for 3 min) have no effect on basal synaptic transmission in the hippocampal CA1 region. Here, we show that prolonged stimulation blocked LTP by activating extrasynaptic GluN2B receptors via glutamate spillover. In addition, we found that this homeostatic mechanism was absent in slices from the SAP102 knockout, providing evidence for a functional coupling between extrasynaptic GluN2B and the SAP102 scaffold protein. In conclusion, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction via the activation of extrasynaptic GluN2B-containing NMDA receptors. NEW & NOTEWORTHY Although long-term potentiation (LTP) is an attractive model for memory storage, it tends to destabilize neuronal circuits because it drives synapses toward a maximum value. Unless opposed by homeostatic mechanisms operating through negative feedback rules, cumulative LTP could render synapses unable to encode additional information. In this study, we uncovered a rapid homeostatic mechanism that antagonizes LTP induction under conditions of prolonged synaptic stimulation via the activation of an extrasynaptic GluN2B-SAP102 complex.

Pyramidal neuron conductance state gates spike-timing-dependent plasticity.

Neocortical neurons in vivo process each of their individual inputs in the context of ongoing synaptic background activity, produced by the thousands of presynaptic partners a typical neuron has. Previous work has shown that background activity affects multiple aspects of neuronal and network function. However, its effect on the induction of spike-timing dependent plasticity (STDP) is not clear. Here we report that injections of simulated background conductances (produced by a dynamic-clamp system) into pyramidal cells in rat brain slices selectively reduced the magnitude of timing-dependent synaptic potentiation while leaving the magnitude of timing-dependent synaptic depression unchanged. The conductance-dependent suppression also sharpened the STDP curve, with reliable synaptic potentiation induced only when EPSPs and action potentials (APs) were paired within 8 ms of each other. Dual somatic and dendritic patch recordings suggested that the deficit in synaptic potentiation arose from shunting of dendritic EPSPs and APs. Using a biophysically detailed computational model, we were not only able to replicate the conductance-dependent shunting of dendritic potentials, but show that synaptic background can truncate calcium dynamics within dendritic spines in a way that affects potentiation more strongly than depression. This conductance-dependent regulation of synaptic plasticity may constitute a novel homeostatic mechanism that can prevent the runaway synaptic potentiation to which Hebbian networks are vulnerable.

An Alternative Pin1 Binding and Isomerization Site in the N-Terminus Domain of PSD-95.

Phosphorylation-dependent peptidyl-prolyl cis-trans isomerization plays key roles in cell cycle progression, the pathogenesis of cancer, and age-related neurodegeneration. Most of our knowledge about the role of phosphorylation-dependent peptidyl-prolyl cis-trans isomerization and the enzyme catalyzing this reaction, the peptidyl-prolyl isomerase (Pin1), is largely limited to proteins not present in neurons. Only a handful of examples have shown that phosphorylation-dependent peptidyl-prolyl cis-trans isomerization, Pin1 binding, or Pin1-mediated peptidyl-prolyl cis-trans isomerization regulate proteins present at excitatory synapses. In this work, I confirm previous findings showing that Pin1 binds postsynaptic density protein-95 (PSD-95) and identify an alternative binding site in the phosphorylated N-terminus of the PSD-95. Pin1 associates via its WW domain with phosphorylated threonine (T19) and serine (S25) in the N-terminus domain of PSD-95 and this association alters the local conformation of PSD-95. Most importantly, I show that proline-directed phosphorylation of the N-terminus domain of PSD-95 alters the local conformation of this region. Therefore, proline-directed phosphorylation of the N-terminus of PSD-95, Pin1 association, and peptidyl-prolyl cis-trans isomerization may all play a role in excitatory synaptic function and synapse development.

Pin1 Binding to Phosphorylated PSD-95 Regulates the Number of Functional Excitatory Synapses.

The post-synaptic density protein 95 (PSD-95) plays a central role in excitatory synapse development and synaptic plasticity. Phosphorylation of the N-terminus of PSD-95 at threonine 19 (T19) and serine 25 (S25) decreases PSD-95 stability at synapses; however, a molecular mechanism linking PSD-95 phosphorylation to altered synaptic stability is lacking. Here, we show that phosphorylation of T19/S25 recruits the phosphorylation-dependent peptidyl-prolyl cis-trans isomerase (Pin1) and reduces the palmitoylation of Cysteine 3 and Cysteine 5 in PSD-95. This reduction in PSD-95 palmitoylation accounts for the observed loss in the number of dendritic PSD-95 clusters, the increased AMPAR mobility, and the decreased number of functional excitatory synapses. We find the effects of Pin1 overexpression were all rescued by manipulations aimed at increasing the levels of PSD-95 palmitoylation. Therefore, Pin1 is a key signaling molecule that regulates the stability of excitatory synapses and may participate in the destabilization of PSD-95 following the induction of synaptic plasticity.

Synapse-associated protein 102/dlgh3 couples the NMDA receptor to specific plasticity pathways and learning strategies.

Understanding the mechanisms whereby information encoded within patterns of action potentials is deciphered by neurons is central to cognitive psychology. The multiprotein complexes formed by NMDA receptors linked to synaptic membrane-associated guanylate kinase (MAGUK) proteins including synapse-associated protein 102 (SAP102) and other associated proteins are instrumental in these processes. Although humans with mutations in SAP102 show mental retardation, the physiological and biochemical mechanisms involved are unknown. Using SAP102 knock-out mice, we found specific impairments in synaptic plasticity induced by selective frequencies of stimulation that also required extracellular signal-regulated kinase signaling. This was paralleled by inflexibility and impairment in spatial learning. Improvement in spatial learning performance occurred with extra training despite continued use of a suboptimal search strategy, and, in a separate nonspatial task, the mutants again deployed a different strategy. Double-mutant analysis of postsynaptic density-95 and SAP102 mutants indicate overlapping and specific functions of the two MAGUKs. These in vivo data support the model that specific MAGUK proteins couple the NMDA receptor to distinct downstream signaling pathways. This provides a mechanism for discriminating patterns of synaptic activity that lead to long-lasting changes in synaptic strength as well as distinct aspects of cognition in the mammalian nervous system.

NMDA receptor activation dephosphorylates AMPA receptor glutamate receptor 1 subunits at threonine 840.

Phosphorylation-dependent changes in AMPA receptor function have a crucial role in activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although three previously identified phosphorylation sites in AMPA receptor glutamate receptor 1 (GluR1) subunits (S818, S831, and S845) appear to have important roles in LTP and LTD, little is known about the role of other putative phosphorylation sites in GluR1. Here, we describe the characterization of a recently identified phosphorylation site in GluR1 at threonine 840. The results of in vivo and in vitro phosphorylation assays suggest that T840 is not a substrate for protein kinases known to phosphorylate GluR1 at previously identified phosphorylation sites, such as protein kinase A, protein kinase C, and calcium/calmodulin-dependent kinase II. Instead, in vitro phosphorylation assays suggest that T840 is a substrate for p70S6 kinase. Although LTP-inducing patterns of synaptic stimulation had no effect on GluR1 phosphorylation at T840 in the hippocampal CA1 region, bath application of NMDA induced a strong, protein phosphatase 1- and/or 2A-mediated decrease in T840 phosphorylation. Moreover, GluR1 phosphorylation at T840 was transiently decreased by a chemical LTD induction protocol that induced a short-term depression of synaptic strength and persistently decreased by a chemical LTD induction protocol that induced a lasting depression of synaptic transmission. Together, our results show that GluR1 phosphorylation at T840 is regulated by NMDA receptor activation and suggest that decreases in GluR1 phosphorylation at T840 may have a role in LTD.

A Revised View on the Role of Surface AMPAR Mobility in Tuning Synaptic Transmission: Limitations, Tools, and Alternative Views.

Calcium dynamics in presynaptic terminals regulate the response dynamics of most central excitatory synapses. However, this dogma has been challenged by the hypothesis that mobility of the postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid subtype glutamate receptors (AMPAR) plays a role in tuning fast excitatory synaptic transmission. In this review, we reevaluate the factors regulating postsynaptic AMPAR mobility, reassess the modeling parameters, analyze the experimental tools, and end by providing alternative ideas stemming from recent results. In particular, newer methods of labeling AMPARs with small fluorophores in live neurons, combined with super-resolution microscopy and sub-second dynamics, lends support to the idea that AMPARs are primarily within the synapse, are greatly constrained, and have much slower mobility than previously thought. We discuss new experiments which may be necessary to readdress the role of postsynaptic AMPAR mobility in tuning fast excitatory synaptic transmission.

Super-resolution imaging of synaptic and Extra-synaptic AMPA receptors with different-sized fluorescent probes.

Previous studies tracking AMPA receptor (AMPAR) diffusion at synapses observed a large mobile extrasynaptic AMPAR pool. Using super-resolution microscopy, we examined how fluorophore size and photostability affected AMPAR trafficking outside of, and within, post-synaptic densities (PSDs) from rats. Organic fluorescent dyes (≈4 nm), quantum dots, either small (≈10 nm diameter; sQDs) or big (>20 nm; bQDs), were coupled to AMPARs via different-sized linkers. We find that >90% of AMPARs labeled with fluorescent dyes or sQDs were diffusing in confined nanodomains in PSDs, which were stable for 15 min or longer. Less than 10% of sQD-AMPARs were extrasynaptic and highly mobile. In contrast, 5-10% of bQD-AMPARs were in PSDs and 90-95% were extrasynaptic as previously observed. Contrary to the hypothesis that AMPAR entry is limited by the occupancy of open PSD 'slots', our findings suggest that AMPARs rapidly enter stable 'nanodomains' in PSDs with lifetime >15 min, and do not accumulate in extrasynaptic membranes.

Long-term potentiation persists in an occult state following mGluR-dependent depotentiation.

Depotentiation, the reversal of long-term potentiation (LTP), can be induced by activation of metabotropic glutamate receptors (mGluRs) or NMDA receptors (NMDARs). Although NMDAR-dependent depotentiation is due to a protein phosphatase-dependent erasure of LTP, the notion that mGluR-dependent depotentiation also involves LTP erasure is controversial. To address this issue we used electrophysiological and biochemical approaches to investigate mGluR-dependent depotentiation in hippocampal slices. Activating group I mGluRs with (R,S)-3,5-dihydroxyphenylglycine (DHPG) induced robust depotentiation in both the CA1 and CA3 regions of hippocampal slices. Western immunoblotting of samples prepared from DHPG-treated slices revealed, however, that activation of group I mGluRs causes a transient increase in phosphorylation of AMPA receptor GluR1 subunits at sites crucial for LTP and under some conditions causes persistent activation of alphaCamKII. The paradoxical ability of DHPG to induce depotentiation while at the same time activating signaling pathways involved in LTP suggests that LTP might not be erased by mGluR-dependent depotentiation. Consistent with this, DHPG-induced depotentiation did not restore the ability of high-frequency stimulation to induce LTP at synapses that had previously undergone saturating levels of LTP. In addition, blocking the expression of DHPG-induced LTD revealed hidden LTP at depotentiated synapses. Our results indicate that LTP and mGluR-dependent LTD can co-exist at excitatory synapses.

The cytochrome c gene proximal enhancer drives activity-dependent reporter gene expression in hippocampal neurons.

The proximal enhancer of the cytochrome c gene (Cycs) contains binding sites for both cAMP response element binding proteins (CREB) and Nuclear Respiratory Factor 1 (NRF1). To investigate how neuronal activity regulates this enhancer region, a lentivirus was constructed in which a short-lived green fluorescent protein (GFP) was placed under the transcriptional control of the Cycs proximal enhancer linked to a synthetic core promoter. Primary hippocampal neurons were infected, and the synaptic strengths of individual neurons were measured by whole-cell patch clamping. On average the amplitude of miniature postsynaptic currents (mEPSCs) was higher in brighter GFP(+) neurons, while the frequency of mEPSCs was not significantly different. Inhibiting neural activity by applying a GABAA receptor agonist increased GFP expression in most neurons, which persisted after homeostatic synaptic scaling as evidenced by a decrease in the amplitude and frequency of mEPSCs. Removing the CREB binding sites revealed that calcium influx through L-type channels and NMDA receptors, and ERK1/2 activation played a role in NRF1-mediated transcription. CREB and NRF1, therefore, combine to regulate transcription of Cycs in response to changing neural activity.

Phosphorylation of proteins involved in activity-dependent forms of synaptic plasticity is altered in hippocampal slices maintained in vitro.

The acute hippocampal slice preparation has been widely used to study the cellular mechanisms underlying activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD). Although protein phosphorylation has a key role in LTP and LTD, little is known about how protein phosphorylation might be altered in hippocampal slices maintained in vitro. To begin to address this issue, we examined the effects of slicing and in vitro maintenance on phosphorylation of six proteins involved in LTP and/or LTD. We found that AMPA receptor (AMPAR) glutamate receptor 1 (GluR1) subunits are persistently dephosphorylated in slices maintained in vitro for up to 8 h. alpha calcium/calmodulin-dependent kinase II (alphaCamKII) was also strongly dephosphorylated during the first 3 h in vitro but thereafter recovered to near control levels. In contrast, phosphorylation of the extracellular signal-regulated kinase ERK2, the ERK kinase MEK, proline-rich tyrosine kinase 2 (Pyk2), and Src family kinases was significantly, but transiently, increased. Electrophysiological experiments revealed that the induction of LTD by low-frequency synaptic stimulation was sensitive to time in vitro. These findings indicate that phosphorylation of proteins involved in N-methyl-D-aspartate (NMDA) receptor-dependent forms of synaptic plasticity is altered in hippocampal slices and suggest that some of these changes can significantly influence the induction of LTD.