Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2.

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5-mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2-ERK1/2 interactions.

A stochastic model of hippocampal synaptic plasticity with geometrical readout of enzyme dynamics.

Discovering the rules of synaptic plasticity is an important step for understanding brain learning. Existing plasticity models are either (1) top-down and interpretable, but not flexible enough to account for experimental data, or (2) bottom-up and biologically realistic, but too intricate to interpret and hard to fit to data. To avoid the shortcomings of these approaches, we present a new plasticity rule based on a geometrical readout mechanism that flexibly maps synaptic enzyme dynamics to predict plasticity outcomes. We apply this readout to a multi-timescale model of hippocampal synaptic plasticity induction that includes electrical dynamics, calcium, CaMKII and calcineurin, and accurate representation of intrinsic noise sources. Using a single set of model parameters, we demonstrate the robustness of this plasticity rule by reproducing nine published ex vivo experiments covering various spike-timing and frequency-dependent plasticity induction protocols, animal ages, and experimental conditions. Our model also predicts that in vivo-like spike timing irregularity strongly shapes plasticity outcome. This geometrical readout modelling approach can be readily applied to other excitatory or inhibitory synapses to discover their synaptic plasticity rules.

Neurotrophin receptor activation rescues cognitive and synaptic abnormalities caused by hemizygosity of the psychiatric risk gene Cacna1c.

Genetic variation in CACNA1C, which encodes the alpha-1 subunit of CaV1.2 L-type voltage-gated calcium channels, is strongly linked to risk for psychiatric disorders including schizophrenia and bipolar disorder. To translate genetics to neurobiological mechanisms and rational therapeutic targets, we investigated the impact of mutations of one copy of Cacna1c on rat cognitive, synaptic and circuit phenotypes implicated by patient studies. We show that rats hemizygous for Cacna1c harbour marked impairments in learning to disregard non-salient stimuli, a behavioural change previously associated with psychosis. This behavioural deficit is accompanied by dys-coordinated network oscillations during learning, pathway-selective disruption of hippocampal synaptic plasticity, attenuated Ca2+ signalling in dendritic spines and decreased signalling through the Extracellular-signal Regulated Kinase (ERK) pathway. Activation of the ERK pathway by a small-molecule agonist of TrkB/TrkC neurotrophin receptors rescued both behavioural and synaptic plasticity deficits in Cacna1c+/- rats. These results map a route through which genetic variation in CACNA1C can disrupt experience-dependent synaptic signalling and circuit activity, culminating in cognitive alterations associated with psychiatric disorders. Our findings highlight targeted activation of neurotrophin signalling pathways with BDNF mimetic drugs as a genetically informed therapeutic approach for rescuing behavioural abnormalities in psychiatric disorder.

Convergent Metabotropic Signaling Pathways Inhibit SK Channels to Promote Synaptic Plasticity in the Hippocampus.

Hebbian synaptic plasticity at hippocampal Schaffer collateral synapses is tightly regulated by postsynaptic small conductance (SK) channels that restrict NMDA receptor activity. SK channels are themselves modulated by G-protein-coupled signaling pathways, but it is not clear under what conditions these are activated to enable synaptic plasticity. Here, we show that muscarinic M1 receptor (M1R) and type 1 metabotropic glutamate receptor (mGluR1) signaling pathways, which are known to inhibit SK channels and thereby disinhibit NMDA receptors, converge to facilitate spine calcium transients during the induction of long-term potentiation (LTP) at hippocampal Schaffer collateral synapses onto CA1 pyramidal neurons of male rats. Furthermore, mGluR1 activation is required for LTP induced by reactivated place-cell firing patterns that occur in sharp-wave ripple events during rest or sleep. In contrast, M1R activation is required for LTP induced by place-cell firing patterns during exploration. Thus, we describe a common mechanism that enables synaptic plasticity during both encoding and consolidation of memories within hippocampal circuits.SIGNIFICANCE STATEMENT Memory ensembles in the hippocampus are formed during active exploration and consolidated during rest or sleep. These two distinct phases each require strengthening of synaptic connections by long-term potentiation (LTP). The neuronal activity patterns in each phase are very different, which makes it hard to map generalized rules for LTP induction onto both formation and consolidation phases. In this study, we show that inhibition of postsynaptic SK channels is a common necessary feature of LTP induction and that SK channel inhibition is achieved by separate but convergent metabotropic signaling pathways. Thus, we reveal a common mechanism for enabling LTP under distinct behavioral conditions.

Neuromodulation of the Feedforward Dentate Gyrus-CA3 Microcircuit.

The feedforward dentate gyrus-CA3 microcircuit in the hippocampus is thought to activate ensembles of CA3 pyramidal cells and interneurons to encode and retrieve episodic memories. The creation of these CA3 ensembles depends on neuromodulatory input and synaptic plasticity within this microcircuit. Here we review the mechanisms by which the neuromodulators aceylcholine, noradrenaline, dopamine, and serotonin reconfigure this microcircuit and thereby infer the net effect of these modulators on the processes of episodic memory encoding and retrieval.

Coordinated activation of distinct Ca(2+) sources and metabotropic glutamate receptors encodes Hebbian synaptic plasticity.

At glutamatergic synapses, induction of associative synaptic plasticity requires time-correlated presynaptic and postsynaptic spikes to activate postsynaptic NMDA receptors (NMDARs). The magnitudes of the ensuing Ca2+ transients within dendritic spines are thought to determine the amplitude and direction of synaptic change. In contrast, we show that at mature hippocampal Schaffer collateral synapses the magnitudes of Ca2+ transients during plasticity induction do not match this rule. Indeed, LTP induced by time-correlated pre- and postsynaptic spikes instead requires the sequential activation of NMDARs followed by voltage-sensitive Ca2+ channels within dendritic spines. Furthermore, LTP requires inhibition of SK channels by mGluR1, which removes a negative feedback loop that constitutively regulates NMDARs. Therefore, rather than being controlled simply by the magnitude of the postsynaptic calcium rise, LTP induction requires the coordinated activation of distinct sources of Ca2+ and mGluR1-dependent facilitation of NMDAR function.

Clathrin-independent trafficking of AMPA receptors.

Membrane trafficking of AMPA receptors (AMPARs) is critical for neuronal function and plasticity. Although rapid forms of AMPAR internalization during long-term depression (LTD) require clathrin and dynamin, the mechanisms governing constitutive AMPAR turnover and internalization of AMPARs during slow homeostatic forms of synaptic plasticity remain unexplored. Here, we show that, in contrast to LTD, constitutive AMPAR internalization and homeostatic AMPAR downscaling in rat neurons do not require dynamin or clathrin function. Instead, constitutive AMPAR trafficking is blocked by a Rac1 inhibitor and is regulated by a dynamic nonstructural pool of F-actin. Our findings reveal a novel role for neuronal clathrin-independent endocytosis controlled by actin dynamics and suggest that the interplay between different modes of receptor endocytosis provides for segregation between distinct modes of neuronal plasticity.

Wavelet transform-based de-noising for two-photon imaging of synaptic Ca2+ transients.

Postsynaptic Ca(2+) transients triggered by neurotransmission at excitatory synapses are a key signaling step for the induction of synaptic plasticity and are typically recorded in tissue slices using two-photon fluorescence imaging with Ca(2+)-sensitive dyes. The signals generated are small with very low peak signal/noise ratios (pSNRs) that make detailed analysis problematic. Here, we implement a wavelet-based de-noising algorithm (PURE-LET) to enhance signal/noise ratio for Ca(2+) fluorescence transients evoked by single synaptic events under physiological conditions. Using simulated Ca(2+) transients with defined noise levels, we analyzed the ability of the PURE-LET algorithm to retrieve the underlying signal. Fitting single Ca(2+) transients with an exponential rise and decay model revealed a distortion of τ(rise) but improved accuracy and reliability of τ(decay) and peak amplitude after PURE-LET de-noising compared to raw signals. The PURE-LET de-noising algorithm also provided a ∼30-dB gain in pSNR compared to ∼16-dB pSNR gain after an optimized binomial filter. The higher pSNR provided by PURE-LET de-noising increased discrimination accuracy between successes and failures of synaptic transmission as measured by the occurrence of synaptic Ca(2+) transients by ∼20% relative to an optimized binomial filter. Furthermore, in comparison to binomial filter, no optimization of PURE-LET de-noising was required for reducing arbitrary bias. In conclusion, the de-noising of fluorescent Ca(2+) transients using PURE-LET enhances detection and characterization of Ca(2+) responses at central excitatory synapses.

CaMKII triggers the diffusional trapping of surface AMPARs through phosphorylation of stargazin.

The Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is critically required for the synaptic recruitment of AMPA-type glutamate receptors (AMPARs) during both development and plasticity. However, the underlying mechanism is unknown. Using single-particle tracking of AMPARs, we show that CaMKII activation and postsynaptic translocation induce the synaptic trapping of AMPARs diffusing in the membrane. AMPAR immobilization requires both phosphorylation of the auxiliary subunit Stargazin and its binding to PDZ domain scaffolds. It does not depend on the PDZ binding domain of GluA1 AMPAR subunit nor its phosphorylation at Ser831. Finally, CaMKII-dependent AMPAR immobilization regulates short-term plasticity. Thus, NMDA-dependent Ca(2+) influx in the post-synapse triggers a CaMKII- and Stargazin-dependent decrease in AMPAR diffusional exchange at synapses that controls synaptic function.

Behavioral deficits and subregion-specific suppression of LTP in mice expressing a population of mutant NMDA receptors throughout the hippocampus.

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.

The glutamate receptor 2 subunit controls post-synaptic density complexity and spine shape in the dentate gyrus.

In adult brain the majority of AMPA glutamate receptor (GluR) subunits contain GluR2. In knock-out (KO) mice the absence of GluR2 results in consequences for synaptic plasticity including cognitive impairments. Here the morphology of dendritic spines and their synaptic contacts was analysed via three-dimensional reconstruction of serial electron micrographs from dentate gyrus (DG) of adult wild type (WT) and GluR2 KO mice. Pre-embedding immunocytochemical staining was used to examine the distribution and subcellular localization of AMPA receptor GluR1 and N-methyl-D-aspartate receptor NR1 subunits. There were no significant changes in synapse density in the DG of GluR2 KO compared with WT mice. However, in GluR2 KO mice there was a significant decrease in the percentage of synapses on mushroom spines but an increase in synapses on thin spines. There was also a large decrease in the proportion of synapses with complex perforated/segmented post-synaptic densities (PSDs) (25 vs. 78% in WT) but an increase in synapses with macular PSDs (75 vs. 22%). These data were coupled in GluR2 KO mice with significant decreases in volume and surface area of mushroom spines and their PSDs. In both GluR2 KO and WT mice, NR1 and GluR1 receptors were present in dendrites and spines but there was a significant reduction in NR1 labelling of spine membranes and cytoplasm in GluR2 KO mice, and a small decrease in GluR1 immunolabelling in membranes and cytoplasm of spines in GluR2 KO compared with WT mice. Our data demonstrate that the absence of GluR2 has a significant effect on both DG synapse and spine cytoarchitecture and the expression of NR1 receptors.

CaMKII translocation requires local NMDA receptor-mediated Ca2+ signaling.

Excitatory synaptic transmission and plasticity are critically modulated by N-methyl-D-aspartate receptors (NMDARs). Activation of NMDARs elevates intracellular Ca(2+) affecting several downstream signaling pathways that involve Ca(2+)/calmodulin-dependent protein kinase II (CaMKII). Importantly, NMDAR activation triggers CaMKII translocation to synaptic sites. NMDAR activation failed to induce Ca(2+) responses in hippocampal neurons lacking the mandatory NMDAR subunit NR1, and no EGFP-CaMKIIalpha translocation was observed. In cells solely expressing Ca(2+)-impermeable NMDARs containing NR1(N598R)-mutant subunits, prolonged NMDA application elevated internal Ca(2+) to the same degree as in wild-type controls, yet failed to translocate CaMKIIalpha. Brief local NMDA application evoked smaller Ca(2+) transients in dendritic spines of mutant compared to wild-type cells. CaMKIIalpha mutants that increase binding to synaptic sites, namely CaMKII-T286D and CaMKII-TT305/306VA, rescued the translocation in NR1(N598R) cells in a glutamate receptor-subtype-specific manner. We conclude that CaMKII translocation requires Ca(2+) entry directly through NMDARs, rather than other Ca(2+) sources activated by NMDARs. Together with the requirement for activated, possibly ligand-bound, NMDARs as CaMKII binding partners, this suggests that synaptic CaMKII accumulation is an input-specific signaling event.

Subcellular localisation of recombinant alpha- and gamma-synuclein.

alpha-Synuclein, a protein implicated in neurodegenerative diseases and of elusive physiological function owes its name to an observed presence in presynaptic and nuclear compartments. However, its nuclear localisation has remained controversial. We expressed synuclein-eGFP fusion proteins in organotypic rat hippocampal slice cultures and murine hippocampal primary neurons using a Sindbis virus expression system. Recombinant full-length alpha-synuclein accumulated in presynaptic locations, mimicking its native distribution. Expression of deletion mutant alpha-synuclein revealed that presynaptic targeting depended on the presence of its N-terminal and core region. This domain also causes nuclear exclusion of the alpha-synuclein fusion protein. In contrast, the C-terminal domain of alpha-synuclein directs fusion proteins into the nuclear compartment. The related protein gamma-synuclein contains a similar N-terminal and core domain as alpha-synuclein. However, gamma-synuclein lacks a C-terminal domain that causes nuclear localisation of the fusion protein, suggesting that the two synucleins might have different roles relating to the cell nucleus.

Absence of Whisker-related pattern formation in mice with NMDA receptors lacking coincidence detection properties and calcium signaling.

Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+ impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement.