EphB-mediated degradation of the RhoA GEF Ephexin5 relieves a developmental brake on excitatory synapse formation.

The mechanisms that promote excitatory synapse formation and maturation have been extensively studied. However, the molecular events that limit excitatory synapse development so that synapses form at the right time and place and in the correct numbers are less well understood. We have identified a RhoA guanine nucleotide exchange factor, Ephexin5, which negatively regulates excitatory synapse development until EphrinB binding to the EphB receptor tyrosine kinase triggers Ephexin5 phosphorylation, ubiquitination, and degradation. The degradation of Ephexin5 promotes EphB-dependent excitatory synapse development and is mediated by Ube3A, a ubiquitin ligase that is mutated in the human cognitive disorder Angelman syndrome and duplicated in some forms of Autism Spectrum Disorders (ASDs). These findings suggest that aberrant EphB/Ephexin5 signaling during the development of synapses may contribute to the abnormal cognitive function that occurs in Angelman syndrome and, possibly, ASDs.

Acute Rift Valley fever virus infection induces inflammatory cytokines and cell death in ex vivo rat brain slice culture.

Rift Valley fever virus (RVFV) is an emerging arboviral disease with pandemic potential. While infection is often self-limiting, a subset of individuals may develop late-onset encephalitis, accounting for up to 20 % of severe cases. Importantly, individuals displaying neurologic disease have up to a 53 % case fatality rate, yet the neuropathogenesis of RVFV infection remains understudied. In this study, we evaluated whether ex vivo postnatal rat brain slice cultures (BSCs) could be used to evaluate RVFV infection in the central nervous system. BSCs mounted an inflammatory response after slicing, which resolved over time, and they were viable in culture for at least 12 days. Infection of rat BSCs with pathogenic RVFV strain ZH501 induced tissue damage and apoptosis over 48 h. Viral replication in BSCs reached up to 1×107 p.f.u. equivalents/ml, depending on inoculation dose. Confocal immunofluorescent microscopy of cleared slices confirmed direct infection of neurons as well as activation of microglia and astrocytes. Further, RVFV-infected rat BSCs produced antiviral cytokines and chemokines, including MCP-1 and GRO/KC. This study demonstrates that rat BSCs support replication of RVFV for ex vivo studies of neuropathogenesis. This allows for continued and complementary investigation into RVFV infection in an ex vivo postnatal brain slice culture format.

A Kalirin missense mutation enhances dendritic RhoA signaling and leads to regression of cortical dendritic arbors across development.

Normally, dendritic size is established prior to adolescence and then remains relatively constant into adulthood due to a homeostatic balance between growth and retraction pathways. However, schizophrenia is characterized by accelerated reductions of cerebral cortex gray matter volume and onset of clinical symptoms during adolescence, with reductions in layer 3 pyramidal neuron dendritic length, complexity, and spine density identified in multiple cortical regions postmortem. Nogo receptor 1 (NGR1) activation of the GTPase RhoA is a major pathway restricting dendritic growth in the cerebral cortex. We show that the NGR1 pathway is stimulated by OMGp and requires the Rho guanine nucleotide exchange factor Kalirin-9 (KAL9). Using a genetically encoded RhoA sensor, we demonstrate that a naturally occurring missense mutation in Kalrn, KAL-PT, that was identified in a schizophrenia cohort, confers enhanced RhoA activitation in neuronal dendrites compared to wild-type KAL. In mice containing this missense mutation at the endogenous locus, there is an adolescent-onset reduction in dendritic length and complexity of layer 3 pyramidal neurons in the primary auditory cortex. Spine density per unit length of dendrite is unaffected. Early adult mice with these structural deficits exhibited impaired detection of short gap durations. These findings provide a neuropsychiatric model of disease capturing how a mild genetic vulnerability may interact with normal developmental processes such that pathology only emerges around adolescence. This interplay between genetic susceptibility and normal adolescent development, both of which possess inherent individual variability, may contribute to heterogeneity seen in phenotypes in human neuropsychiatric disease.

Endogenous PTEN-Induced Kinase 1 Regulates Dendritic Architecture and Spinogenesis.

Mutations in PTEN-induced kinase 1 (PINK1) contribute to autosomal recessive Parkinson's disease with cognitive and neuropsychiatric comorbidities. Disturbances in dendritic and spine architecture are hallmarks of neurodegenerative and neuropsychiatric conditions, but little is known of the impact of PINK1 on these structures. We used Pink1 -/- mice to study the role of endogenous PINK1 in regulating dendritic architecture, spine density, and spine maturation. Pink1 -/- cortical neurons of unknown sex showed decreased dendritic arborization, affecting both apical and basal arbors. Dendritic simplification in Pink1 -/- neurons was primarily driven by diminished branching with smaller effects on branch lengths. Pink1 -/- neurons showed reduced spine density with a shift in morphology to favor filopodia at the expense of mushroom spines. Electrophysiology revealed significant reductions in miniature EPSC (mEPSC) frequency in Pink1 -/- neurons, consistent with the observation of decreased spine numbers. Transfecting with human PINK1 rescued changes in dendritic architecture, in thin, stubby, and mushroom spine densities, and in mEPSC frequency. Diminished spine density was also observed in Golgi-Cox stained adult male Pink1 -/- brains. Western blot study of Pink1 -/- brains of either sex revealed reduced phosphorylation of NSFL1 cofactor p47, an indirect target of PINK1. Transfection of Pink1 -/- neurons with a phosphomimetic p47 plasmid rescued dendritic branching and thin/stubby spine density with a partial rescue of mushroom spines, implicating a role for PINK1-regulated p47 phosphorylation in dendrite and spine development. These findings suggest that PINK1-dependent synaptodendritic alterations may contribute to the risk of cognitive and/or neuropsychiatric pathologies observed in PINK1-mutated families.SIGNIFICANCE STATEMENT Loss of PINK1 function has been implicated in both familial and sporadic neurodegenerative diseases. Yet surprisingly little is known of the impact of PINK1 loss on the fine structure of neurons. Neurons receive excitatory synaptic signals along a complex network of projections that form the dendritic tree, largely at tiny protrusions called dendritic spines. We studied cortical neurons and brain tissues from mice lacking PINK1. We discovered that PINK1 deficiency causes striking simplification of dendritic architecture associated with reduced synaptic input and decreased spine density and maturation. These changes are reversed by reintroducing human PINK1 or one of its downstream mediators into PINK1-deficient mouse neurons, indicating a conserved function, whose loss may contribute to neurodegenerative processes.

Developmental pruning of excitatory synaptic inputs to parvalbumin interneurons in monkey prefrontal cortex.

Working memory requires efficient excitatory drive to parvalbumin-positive (PV) interneurons in the primate dorsolateral prefrontal cortex (DLPFC). Developmental pruning eliminates superfluous excitatory inputs, suggesting that working memory maturation during adolescence requires pruning of excitatory inputs to PV interneurons. Therefore, we tested the hypothesis that excitatory synapses on PV interneurons are pruned during adolescence. The density of excitatory synapses, defined by overlapping vesicular glutamate transporter 1-positive (VGlut1+) and postsynaptic density 95-positive (PSD95+) puncta, on PV interneurons was lower in postpubertal relative to prepubertal monkeys. In contrast, puncta levels of VGlut1 and PSD95 proteins were higher in postpubertal monkeys and positively predicted activity-dependent PV levels, suggesting a greater strength of the remaining synapses after pruning. Because excitatory synapse number on PV interneurons is regulated by erb-b2 receptor tyrosine kinase 4 (ErbB4), whose function is influenced by alternative splicing, we tested the hypothesis that pruning of excitatory synapses on PV interneurons is associated with developmental shifts in ErbB4 expression and/or splicing. Pan-ErbB4 expression did not change, whereas the minor-to-major splice variant ratios increased with age. In cell culture, the major, but not the minor, variant increased excitatory synapse number on PV interneurons and displayed greater kinase activity than the minor variant, suggesting that the effect of ErbB4 signaling in PV interneurons is mediated by alternative splicing. Supporting this interpretation, in monkey DLPFC, higher minor-to-major variant ratios predicted lower PSD95+ puncta density on PV interneurons. Together, our findings suggest that ErbB4 splicing may regulate the pruning of excitatory synapses on PV interneurons during adolescence.

Mitochondrial Calcium Dysregulation Contributes to Dendrite Degeneration Mediated by PD/LBD-Associated LRRK2 Mutants.

Mutations in leucine-rich repeat kinase 2 (LRRK2) contribute to development of late-onset familial Parkinson's disease (PD), with clinical features of motor and cognitive dysfunction indistinguishable from sporadic PD. Calcium dysregulation plays an important role in PD pathogenesis, but the mechanisms of neurodegeneration remain unclear. Recent reports indicate enhanced excitatory neurotransmission in cortical neurons expressing mutant LRRK2, which occurs before the well-characterized phenotype of dendritic shortening. As mitochondria play a major role in the rapid buffering of cytosolic calcium, we hypothesized that altered mitochondrial calcium handling contributes to dendritic retraction elicited by the LRRK2-G2019S and -R1441C mutations. In primary mouse cortical neurons, we observed increased depolarization-induced mitochondrial calcium uptake. We found that expression of mutant LRRK2 elicited transcriptional upregulation of the mitochondrial calcium uniporter (MCU) and the mitochondrial calcium uptake 1 protein (MICU1) with no change in levels of the mitochondrial calcium antiporter NCLX. Elevated MCU and MICU1 were also observed in LRRK2-mutated patient fibroblasts, along with increased mitochondrial calcium uptake, and in postmortem brains of sporadic PD/PDD patients of both sexes. Transcriptional upregulation of MCU and MICU1 was caused by activation of the ERK1/2 (MAPK3/1) pathway. Inhibiting ERK1/2 conferred protection against mutant LRRK2-induced neurite shortening. Pharmacological inhibitors or RNAi knockdown of MCU attenuated mitochondrial calcium uptake and dendritic/neuritic shortening elicited by mutant LRRK2, whereas expression of a constitutively active mutant of NCLX that enhances calcium export from mitochondria was neuroprotective. These data suggest that an increased susceptibility to mitochondrial calcium dysregulation contributes to dendritic injury in mutant LRRK2 pathogenesis.SIGNIFICANCE STATEMENT Cognitive dysfunction and dementia are common features of Parkinson's disease (PD), causing significant disability. Mutations in LRRK2 represent the most common known genetic cause of PD. We found that PD-linked LRRK2 mutations increased dendritic and mitochondrial calcium uptake in cortical neurons and familial PD patient fibroblasts, accompanied by increased expression of the mitochondrial calcium transporter MCU. Blocking the ERK1/2-dependent upregulation of MCU conferred protection against mutant LRRK2-elicited dendrite shortening, as did inhibiting MCU-mediated calcium import. Conversely, stimulating the export of calcium from mitochondria was also neuroprotective. These results implicate increased susceptibility to mitochondrial calcium overload in LRRK2-driven neurodegeneration, and suggest possible interventions that may slow the progression of cognitive dysfunction in PD.

Zn(2+) -induced Ca(2+) release via ryanodine receptors triggers calcineurin-dependent redistribution of cortical neuronal Kv2.1 K(+) channels.

KEY POINTS: Increases in intracellular Zn(2+) concentrations are an early, necessary signal for the modulation of Kv2.1 K(+) channel localization and physiological function. Intracellular Zn(2+) -mediated Kv2.1 channel modulation is dependent on calcineurin, a Ca(2+) -activated phosphatase. We show that intracellular Zn(2+) induces a significant increase in ryanodine receptor-dependent cytosolic Ca(2+) transients, which leads to a calcineurin-dependent redistribution of Kv2.1 channels from pre-existing membrane clusters to diffuse localization. As such, the link between Zn(2+) and Ca(2+) signalling in this Kv2.1 modulatory pathway is established. We observe that a sublethal ischaemic preconditioning insult also leads to Kv2.1 redistribution in a ryanodine receptor-dependent fashion. We suggest that Zn(2+) may be an early and ubiquitous signalling molecule mediating Ca(2+) release from the cortical endoplasmic reticulum via ryanodine receptor activation. ABSTRACT: Sublethal injurious stimuli in neurons induce transient increases in free intracellular Zn(2+) that are associated with regulating adaptive responses to subsequent lethal injury, including alterations in the function and localization of the delayed-rectifier potassium channel, Kv2.1. However, the link between intracellular Zn(2+) signalling and the observed changes in Kv2.1 remain undefined. In the present study, utilizing exogenous Zn(2+) treatment, along with a selective Zn(2+) ionophore, we show that transient elevations in intracellular Zn(2+) concentrations are sufficient to induce calcineurin-dependent Kv2.1 channel dispersal in rat cortical neurons in vitro, which is accompanied by a relatively small but significant hyperpolarizing shift in the voltage-gated activation kinetics of the channel. Critically, using a molecularly encoded calcium sensor, we found that the calcineurin-dependent changes in Kv2.1 probably occur as a result of Zn(2+) -induced cytosolic Ca(2+) release via activation of neuronal ryanodine receptors. Finally, we couple this mechanism with an established model for in vitro ischaemic preconditioning and show that Kv2.1 channel modulation in this process is also ryanodine receptor-sensitive. Our results strongly suggest that intracellular Zn(2+) -initiated signalling may represent an early and possibly widespread component of Ca(2+) -dependent processes in neurons.

Cyclin e1 regulates Kv2.1 channel phosphorylation and localization in neuronal ischemia.

Kv2.1 is a major delayed rectifying K(+) channel normally localized to highly phosphorylated somatodendritic clusters in neurons. Excitatory stimuli induce calcineurin-dependent dephosphorylation and dispersal of Kv2.1 clusters, with a concomitant hyperpolarizing shift in the channel's activation kinetics. We showed previously that sublethal ischemia, which renders neurons transiently resistant to excitotoxic cell death, can also induce Zn(2+)-dependent changes in Kv2.1 localization and activation kinetics, suggesting that activity-dependent modifications of Kv2.1 may contribute to cellular adaptive responses to injury. Recently, cyclin-dependent kinase 5 (Cdk5) was shown to phosphorylate Kv2.1, with pharmacological Cdk5 inhibition being sufficient to decluster channels. In another study, cyclin E1 was found to restrict neuronal Cdk5 kinase activity. We show here that cyclin E1 regulates Kv2.1 cellular localization via inhibition of Cdk5 activity. Expression of cyclin E1 in human embryonic kidney cells prevents Cdk5-mediated phosphorylation of Kv2.1, and cyclin E1 overexpression in rat cortical neurons triggers dispersal of Kv2.1 channel clusters. Sublethal ischemia in neurons induces calcineurin-dependent upregulation of cyclin E1 protein expression and cyclin E1-dependent Kv2.1 channel declustering. Importantly, overexpression of cyclin E1 in neurons is sufficient to reduce excitotoxic cell death. These results support a novel role for neuronal cyclin E1 in regulating the phosphorylation status and localization of Kv2.1 channels, a likely component of signaling cascades leading to ischemic preconditioning.

Experimental and computational evidence for an essential role of NaV1.6 in spike initiation at stretch-sensitive colorectal afferent endings.

Stretch-sensitive afferents comprise ∼33% of the pelvic nerve innervation of mouse colorectum, which are activated by colorectal distension and encode visceral nociception. Stretch-sensitive colorectal afferent endings respond tonically to stepped or ramped colorectal stretch, whereas dissociated colorectal dorsal root ganglion neurons generally fail to spike repetitively upon stepped current stimulation. The present study investigated this difference in the neural encoding characteristics between the soma and afferent ending using pharmacological approaches in an in vitro mouse colon-nerve preparation and complementary computational simulations. Immunohistological staining and Western blots revealed the presence of voltage-gated sodium channel (NaV) 1.6 and NaV1.7 at sensory neuronal endings in mouse colorectal tissue. Responses of stretch-sensitive colorectal afferent endings were significantly reduced by targeting NaV1.6 using selective antagonists (µ-conotoxin GIIIa and µ-conotoxin PIIIa) or tetrodotoxin. In contrast, neither selective NaV1.8 (A803467) nor NaV1.7 (ProTX-II) antagonists attenuated afferent responses to stretch. Computational simulation of a colorectal afferent ending that incorporated independent Markov models for NaV1.6 and NaV1.7, respectively, recapitulated the experimental findings, suggesting a necessary role for NaV1.6 in encoding tonic spiking by stretch-sensitive afferents. In addition, computational simulation of a dorsal root ganglion soma showed that, by adding a NaV1.6 conductance, a single-spiking neuron was converted into a tonic spiking one. These results suggest a mechanism/channel to explain the difference in neural encoding characteristics between afferent somata and sensory endings, likely caused by differential expression of ion channels (e.g., NaV1.6) at different parts of the neuron.

Potentiation of BKCa channels by cystic fibrosis transmembrane conductance regulator correctors VX-445 and VX-121.

Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel, ultimately leading to diminished transepithelial anion secretion and mucociliary clearance. CFTR correctors are therapeutics that restore the folding/trafficking of mutated CFTR to the plasma membrane. The large-conductance calcium-activated potassium channel (BKCa, KCa1.1) is also critical for maintaining lung airway surface liquid (ASL) volume. Here, we show that the class 2 (C2) CFTR corrector VX-445 (elexacaftor) induces K+ secretion across WT and F508del CFTR primary human bronchial epithelial cells (HBEs), which was entirely inhibited by the BKCa antagonist paxilline. Similar results were observed with VX-121, a corrector under clinical evaluation. Whole-cell patch-clamp recordings verified that CFTR correctors potentiated BKCa activity from both primary HBEs and HEK cells stably expressing the α subunit (HEK-BK cells). Furthermore, excised patch-clamp recordings from HEK-BK cells verified direct action on the channel and demonstrated a significant increase in open probability. In mouse mesenteric artery, VX-445 induced a paxilline-sensitive vasorelaxation of preconstricted arteries. VX-445 also reduced firing frequency in primary rat hippocampal and cortical neurons. We raise the possibilities that C2 CFTR correctors gain additional clinical benefit by activation of BKCa in the lung yet may lead to adverse events through BKCa activation elsewhere.

Ribosome-Associated Vesicles promote activity-dependent local translation.

Local protein synthesis in axons and dendrites underpins synaptic plasticity. However, the composition of the protein synthesis machinery in distal neuronal processes and the mechanisms for its activity-driven deployment to local translation sites remain unclear. Here, we employed cryo-electron tomography, volume electron microscopy, and live-cell imaging to identify Ribosome-Associated Vesicles (RAVs) as a dynamic platform for moving ribosomes to distal processes. Stimulation via chemically-induced long-term potentiation causes RAV accumulation in distal sites to drive local translation. We also demonstrate activity-driven changes in RAV generation and dynamics in vivo, identifying tubular ER shaping proteins in RAV biogenesis. Together, our work identifies a mechanism for ribosomal delivery to distal sites in neurons to promote activity-dependent local translation.

Ribosome-associated vesicles: A dynamic subcompartment of the endoplasmic reticulum in secretory cells.

The endoplasmic reticulum (ER) is a highly dynamic network of membranes. Here, we combine live-cell microscopy with in situ cryo-electron tomography to directly visualize ER dynamics in several secretory cell types including pancreatic ß-cells and neurons under near-native conditions. Using these imaging approaches, we identify a novel, mobile form of ER, ribosome-associated vesicles (RAVs), found primarily in the cell periphery, which is conserved across different cell types and species. We show that RAVs exist as distinct, highly dynamic structures separate from the intact ER reticular architecture that interact with mitochondria via direct intermembrane contacts. These findings describe a new ER subcompartment within cells.

RNA Binding Antagonizes Neurotoxic Phase Transitions of TDP-43.

TDP-43 proteinopathy is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia where cytoplasmic TDP-43 inclusions are observed within degenerating regions of patient postmortem tissue. The mechanism by which TDP-43 aggregates has remained elusive due to technological limitations, which prevent the analysis of specific TDP-43 interactions in live cells. We present an optogenetic approach to reliably induce TDP-43 proteinopathy under spatiotemporal control. We show that the formation of pathologically relevant inclusions is driven by aberrant interactions between low-complexity domains of TDP-43 that are antagonized by RNA binding. Although stress granules are hypothesized to be a conduit for seeding TDP-43 proteinopathy, we demonstrate pathological inclusions outside these RNA-rich structures. Furthermore, we show that aberrant phase transitions of cytoplasmic TDP-43 are neurotoxic and that treatment with oligonucleotides composed of TDP-43 target sequences prevent inclusions and rescue neurotoxicity. Collectively, these studies provide insight into the mechanisms that underlie TDP-43 proteinopathy and present a potential avenue for therapeutic intervention.

Kalirin reduction rescues psychosis-associated behavioral deficits in APPswe/PSEN1dE9 transgenic mice.

Psychosis in Alzheimer's disease (AD+P) represents a distinct clinical and neurobiological AD phenotype and is associated with more rapid cognitive decline, higher rates of abnormal behaviors, and increased caregiver burden compared with AD without psychosis. On a molecular level, AD+P is associated with greater reductions in the protein kalirin, a guanine exchange factor which has also been linked to the psychotic disease, schizophrenia. In this study, we sought to determine the molecular and behavioral consequences of kalirin reduction in APPswe/PSEN1dE9 mice. We evaluated mice with and without kalirin reduction during tasks measuring psychosis-associated behaviors and spatial memory. We found that kalirin reduction in APPswe/PSEN1dE9 mice significantly attenuated psychosis-associated behavior at 12 months of age without changing spatial memory performance. The 12-month-old APPswe/PSEN1dE9 mice with reduced kalirin levels also had increased levels of the active, phosphorylated forms of p21 protein (Cdc42/Rac)-activated kinases (PAKs), which function in signaling pathways for maintenance of dendritic spine density, morphology, and function.

Selective Loss of Smaller Spines in Schizophrenia.

OBJECTIVE: Decreased density of dendritic spines in adult schizophrenia subjects has been hypothesized to result from increased pruning of excess synapses in adolescence. In vivo imaging studies have confirmed that synaptic pruning is largely driven by the loss of large or mature synapses. Thus, increased pruning throughout adolescence would likely result in a deficit of large spines in adulthood. Here, the authors examined the density and volume of dendritic spines in deep layer 3 of the auditory cortex of 20 schizophrenia and 20 matched comparison subjects as well as aberrant voltage-gated calcium channel subunit protein expression linked to spine loss. METHOD: Primary auditory cortex deep layer 3 spine density and volume was assessed in 20 pairs of schizophrenia and matched comparison subjects in an initial and replication cohort (12 and eight pairs) by immunohistochemistry-confocal microscopy. Targeted mass spectrometry was used to quantify postsynaptic density and voltage-gated calcium channel protein expression. The effect of increased voltage-gated calcium channel subunit protein expression on spine density and volume was assessed in primary rat neuronal culture. RESULTS: Only the smallest spines are lost in deep layer 3 of the primary auditory cortex in subjects with schizophrenia, while larger spines are retained. Levels of the tryptic peptide ALFDFLK, found in the schizophrenia risk gene CACNB4, are inversely correlated with the density of smaller, but not larger, spines in schizophrenia subjects. Consistent with this observation, CACNB4 overexpression resulted in a lower density of smaller spines in primary neuronal cultures. CONCLUSIONS: These findings require a rethinking of the overpruning hypothesis, demonstrate a link between small spine loss and a schizophrenia risk gene, and should spur more in-depth investigations of the mechanisms that govern new or small spine generation and stabilization under normal conditions as well as how this process is impaired in schizophrenia.

Amyloid Beta Peptides Block New Synapse Assembly by Nogo Receptor-Mediated Inhibition of T-Type Calcium Channels.

Compelling evidence links amyloid beta (Aß) peptide accumulation in the brains of Alzheimer's disease (AD) patients with the emergence of learning and memory deficits, yet a clear understanding of the events that drive this synaptic pathology are lacking. We present evidence that neurons exposed to Aß are unable to form new synapses, resulting in learning deficits in vivo. We demonstrate the Nogo receptor family (NgR1-3) acts as Aß receptors mediating an inhibition of synapse assembly, plasticity, and learning. Live imaging studies reveal Aß activates NgRs on the dendritic shaft of neurons, triggering an inhibition of calcium signaling. We define T-type calcium channels as a target of Aß-NgR signaling, mediating Aß's inhibitory effects on calcium, synapse assembly, plasticity, and learning. These studies highlight deficits in new synapse assembly as a potential initiator of cognitive pathology in AD, and pinpoint calcium dysregulation mediated by NgRs and T-type channels as key components. VIDEO ABSTRACT.

The nogo receptor family restricts synapse number in the developing hippocampus.

Neuronal development is characterized by a period of exuberant synaptic growth that is well studied. However, the mechanisms that restrict this process are less clear. Here we demonstrate that glycosylphosphatidylinositol-anchored cell-surface receptors of the Nogo Receptor family (NgR1, NgR2, and NgR3) restrict excitatory synapse formation. Loss of any one of the NgRs results in an increase in synapse number in vitro, whereas loss of all three is necessary for abnormally elevated synaptogenesis in vivo. We show that NgR1 inhibits the formation of new synapses in the postsynaptic neuron by signaling through the coreceptor TROY and RhoA. The NgR family is downregulated by neuronal activity, a response that may limit NgR function and facilitate activity-dependent synapse development. These findings suggest that NgR1, a receptor previously shown to restrict axon growth in the adult, also functions in the dendrite as a barrier that limits excitatory synapse number during brain development.

Cyclin E constrains Cdk5 activity to regulate synaptic plasticity and memory formation.

Cyclin E is a component of the core cell cycle machinery, and it drives cell proliferation by regulating entry and progression of cells through the DNA synthesis phase. Cyclin E expression is normally restricted to proliferating cells. However, high levels of cyclin E are expressed in the adult brain. The function of cyclin E in quiescent, postmitotic nervous system remains unknown. Here we use a combination of in vivo quantitative proteomics and analyses of cyclin E knockout mice to demonstrate that in terminally differentiated neurons cyclin E forms complexes with Cdk5 and controls synapse function by restraining Cdk5 activity. Ablation of cyclin E led to a decreased number of synapses, reduced number and volume of dendritic spines, and resulted in impaired synaptic plasticity and memory formation in cyclin E-deficient animals. These results reveal a cell cycle-independent role for a core cell cycle protein, cyclin E, in synapse function and memory.