Repopulating Microglia Promote Brain Repair in an IL-6-Dependent Manner.

Cognitive dysfunction and reactive microglia are hallmarks of traumatic brain injury (TBI), yet whether these cells contribute to cognitive deficits and secondary inflammatory pathology remains poorly understood. Here, we show that removal of microglia from the mouse brain has little effect on the outcome of TBI, but inducing the turnover of these cells through either pharmacologic or genetic approaches can yield a neuroprotective microglial phenotype that profoundly aids recovery. The beneficial effects of these repopulating microglia are critically dependent on interleukin-6 (IL-6) trans-signaling via the soluble IL-6 receptor (IL-6R) and robustly support adult neurogenesis, specifically by augmenting the survival of newborn neurons that directly support cognitive function. We conclude that microglia in the mammalian brain can be manipulated to adopt a neuroprotective and pro-regenerative phenotype that can aid repair and alleviate the cognitive deficits arising from brain injury.

Representations of Novelty and Familiarity in a Mushroom Body Compartment.

Animals exhibit a behavioral response to novel sensory stimuli about which they have no prior knowledge. We have examined the neural and behavioral correlates of novelty and familiarity in the olfactory system of Drosophila. Novel odors elicit strong activity in output neurons (MBONs) of the α'3 compartment of the mushroom body that is rapidly suppressed upon repeated exposure to the same odor. This transition in neural activity upon familiarization requires odor-evoked activity in the dopaminergic neuron innervating this compartment. Moreover, exposure of a fly to novel odors evokes an alerting response that can also be elicited by optogenetic activation of α'3 MBONs. Silencing these MBONs eliminates the alerting behavior. These data suggest that the α'3 compartment plays a causal role in the behavioral response to novel and familiar stimuli as a consequence of dopamine-mediated plasticity at the Kenyon cell-MBONα'3 synapse.

Considering Organismal Physiology in Laboratory Studies of Rodent Behavior.

Any experiment conducted in a rodent laboratory is done so against the backdrop of each animal's physiological state at the time of the experiment. This physiological state can be the product of multiple factors, both internal (e.g., animal sex, strain, hormone cycles, or circadian rhythms) and external (e.g., housing conditions, social status, and light/dark phases). Each of these factors has the potential to influence experimental outcomes, either independently or via interactions with others, and yet there is little consistency across laboratories in terms of the weight with which they are considered in experimental design. Such discrepancies-both in practice and in reporting-likely contribute to the perception of a reproducibility crisis in the field of behavioral neuroscience. In this review, we discuss how several of these sources of variability can impact outcomes within the realm of common learning and memory paradigms.

The DDHD2-STXBP1 interaction mediates long-term memory via generation of saturated free fatty acids.

The phospholipid and free fatty acid (FFA) composition of neuronal membranes plays a crucial role in learning and memory, but the mechanisms through which neuronal activity affects the brain's lipid landscape remain largely unexplored. The levels of saturated FFAs, particularly of myristic acid (C14:0), strongly increase during neuronal stimulation and memory acquisition, suggesting the involvement of phospholipase A1 (PLA1) activity in synaptic plasticity. Here, we show that genetic ablation of the PLA1 isoform DDHD2 in mice dramatically reduces saturated FFA responses to memory acquisition across the brain. Furthermore, DDHD2 loss also decreases memory performance in reward-based learning and spatial memory models prior to the development of neuromuscular deficits that mirror human spastic paraplegia. Via pulldown-mass spectrometry analyses, we find that DDHD2 binds to the key synaptic protein STXBP1. Using STXBP1/2 knockout neurosecretory cells and a haploinsufficient STXBP1+/- mouse model of human early infantile encephalopathy associated with intellectual disability and motor dysfunction, we show that STXBP1 controls targeting of DDHD2 to the plasma membrane and generation of saturated FFAs in the brain. These findings suggest key roles for DDHD2 and STXBP1 in lipid metabolism and in the processes of synaptic plasticity, learning, and memory.

Reward Contributions to Serotonergic Functions.

The brain serotonin systems participate in numerous aspects of reward processing, although it remains elusive how exactly serotonin signals regulate neural computation and reward-related behavior. The application of optogenetics and imaging techniques during the last decade has provided many insights. Here, we review recent progress on the organization and physiology of the dorsal raphe serotonin neurons and the relationships between their activity and behavioral functions in the context of reward processing. We also discuss several interesting theories on serotonin's function and how these theories may be reconciled by the possibility that serotonin, acting in synergy with coreleased glutamate, tracks and calculates the so-called beneficialness of the current state to guide an animal's behavior in dynamic environments.

Cytoplasmic polyadenylation element binding proteins in development, health, and disease.

The cytoplasmic polyadenylation element binding (CPEB) proteins are sequence-specific mRNA binding proteins that control translation in development, health, and disease. CPEB1, the founding member of this family, has become an important model for illustrating general principles of translational control by cytoplasmic polyadenylation in gametogenesis, cancer etiology, synaptic plasticity, learning, and memory. Although the biological functions of the other members of this protein family in vertebrates are just beginning to emerge, it is already evident that they, too, mediate important processes, such as cancer etiology and higher cognitive function. In Drosophila, the CPEB proteins Orb and Orb2 play key roles in oogenesis and in neuronal function, as do related proteins in Caenorhabditis elegans and Aplysia. We review the biochemical features of the CPEB proteins, discuss their activities in several biological systems, and illustrate how understanding CPEB activity in model organisms has an important impact on neurological disease.

Autism-Misregulated eIF4G Microexons Control Synaptic Translation and Higher Order Cognitive Functions.

Microexons represent the most highly conserved class of alternative splicing, yet their functions are poorly understood. Here, we focus on closely related neuronal microexons overlapping prion-like domains in the translation initiation factors, eIF4G1 and eIF4G3, the splicing of which is activity dependent and frequently disrupted in autism. CRISPR-Cas9 deletion of these microexons selectively upregulates synaptic proteins that control neuronal activity and plasticity and further triggers a gene expression program mirroring that of activated neurons. Mice lacking the Eif4g1 microexon display social behavior, learning, and memory deficits, accompanied by altered hippocampal synaptic plasticity. We provide evidence that the eIF4G microexons function as a translational brake by causing ribosome stalling, through their propensity to promote the coalescence of cytoplasmic granule components associated with translation repression, including the fragile X mental retardation protein FMRP. The results thus reveal an autism-disrupted mechanism by which alternative splicing specializes neuronal translation to control higher order cognitive functioning.

Queuosine-tRNA promotes sex-dependent learning and memory formation by maintaining codon-biased translation elongation speed.

Queuosine (Q) is a modified nucleoside at the wobble position of specific tRNAs. In mammals, queuosinylation is facilitated by queuine uptake from the gut microbiota and is introduced into tRNA by the QTRT1-QTRT2 enzyme complex. By establishing a Qtrt1 knockout mouse model, we discovered that the loss of Q-tRNA leads to learning and memory deficits. Ribo-Seq analysis in the hippocampus of Qtrt1-deficient mice revealed not only stalling of ribosomes on Q-decoded codons, but also a global imbalance in translation elongation speed between codons that engage in weak and strong interactions with their cognate anticodons. While Q-dependent molecular and behavioral phenotypes were identified in both sexes, female mice were affected more severely than males. Proteomics analysis confirmed deregulation of synaptogenesis and neuronal morphology. Together, our findings provide a link between tRNA modification and brain functions and reveal an unexpected role of protein synthesis in sex-dependent cognitive performance.

Arc Oligomerization Is Regulated by CaMKII Phosphorylation of the GAG Domain: An Essential Mechanism for Plasticity and Memory Formation.

Arc is a synaptic protein essential for memory consolidation. Recent studies indicate that Arc originates in evolution from a Ty3-Gypsy retrotransposon GAG domain. The N-lobe of Arc GAG domain acquired a hydrophobic binding pocket in higher vertebrates that is essential for Arc's canonical function to weaken excitatory synapses. Here, we report that Arc GAG also acquired phosphorylation sites that can acutely regulate its synaptic function. CaMKII phosphorylates the N-lobe of the Arc GAG domain and disrupts an interaction surface essential for high-order oligomerization. In Purkinje neurons, CaMKII phosphorylation acutely reverses Arc's synaptic action. Mutant Arc that cannot be phosphorylated by CaMKII enhances metabotropic receptor-dependent depression in the hippocampus but does not alter baseline synaptic transmission or long-term potentiation. Behavioral studies indicate that hippocampus- and amygdala-dependent learning requires Arc GAG domain phosphorylation. These studies provide an atomic model for dynamic and local control of Arc function underlying synaptic plasticity and memory.

The ISR downstream target ATF4 represses long-term memory in a cell type-specific manner.

The integrated stress response (ISR), a pivotal protein homeostasis network, plays a critical role in the formation of long-term memory (LTM). The precise mechanism by which the ISR controls LTM is not well understood. Here, we report insights into how the ISR modulates the mnemonic process by using targeted deletion of the activating transcription factor 4 (ATF4), a key downstream effector of the ISR, in various neuronal and non-neuronal cell types. We found that the removal of ATF4 from forebrain excitatory neurons (but not from inhibitory neurons, cholinergic neurons, or astrocytes) enhances LTM formation. Furthermore, the deletion of ATF4 in excitatory neurons lowers the threshold for the induction of long-term potentiation, a cellular model for LTM. Transcriptomic and proteomic analyses revealed that ATF4 deletion in excitatory neurons leads to upregulation of components of oxidative phosphorylation pathways, which are critical for ATP production. Thus, we conclude that ATF4 functions as a memory repressor selectively within excitatory neurons.

Postnatal expression of the lysine methyltransferase SETD1B is essential for learning and the regulation of neuron-enriched genes.

In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron-specific ChIP-seq and RNA-seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron-specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B-dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function.

Pattern dynamics and stochasticity of the brain rhythms.

Our current understanding of brain rhythms is based on quantifying their instantaneous or time-averaged characteristics. What remains unexplored is the actual structure of the waves-their shapes and patterns over finite timescales. Here, we study brain wave patterning in different physiological contexts using two independent approaches: The first is based on quantifying stochasticity relative to the underlying mean behavior, and the second assesses "orderliness" of the waves' features. The corresponding measures capture the waves' characteristics and abnormal behaviors, such as atypical periodicity or excessive clustering, and demonstrate coupling between the patterns' dynamics and the animal's location, speed, and acceleration. Specifically, we studied patterns of θ, γ, and ripple waves recorded in mice hippocampi and observed speed-modulated changes of the wave's cadence, an antiphase relationship between orderliness and acceleration, as well as spatial selectiveness of patterns. Taken together, our results offer a complementary-mesoscale-perspective on brain wave structure, dynamics, and functionality.

mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory.

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. Here, we report that learning in the contextual fear conditioning paradigm engenders a decrease in eIF2α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories.

Human Presenilin-1 delivered by AAV9 rescues impaired γ-secretase activity, memory deficits, and neurodegeneration in Psen mutant mice.

Mutations in the Presenilin (PSEN1 and PSEN2) genes are the major cause of early-onset familial Alzheimer's disease (FAD). Presenilin (PS) is the catalytic subunit of the γ-secretase complex, which cleaves type I transmembrane proteins, such as Notch and the amyloid precursor protein (APP), and plays an evolutionarily conserved role in the protection of neuronal survival during aging. FAD PSEN1 mutations exhibit impaired γ-secretase activity in cell culture, in vitro, and knockin (KI) mouse brains, and the L435F mutation is the most severe in reducing γ-secretase activity and is located closest to the active site of γ-secretase. Here, we report that introduction of the codon-optimized wild-type human PSEN1 cDNA by adeno-associated virus 9 (AAV9) results in broadly distributed, sustained, low to moderate levels of human PS1 (hPS1) expression and rescues impaired γ-secretase activity in the cerebral cortex of Psen mutant mice either lacking PS or expressing the Psen1 L435F KI allele, as evaluated by endogenous γ-secretase substrates of APP and recombinant γ-secretase products of Notch intracellular domain and Aß peptides. Furthermore, introduction of hPS1 by AAV9 alleviates impairments of synaptic plasticity and learning and memory in Psen mutant mice. Importantly, AAV9 delivery of hPS1 ameliorates neurodegeneration in the cerebral cortex of aged Psen mutant mice, as shown by the reversal of age-dependent loss of cortical neurons and elevated microgliosis and astrogliosis. These results together show that moderate hPS1 expression by AAV9 is sufficient to rescue impaired γ-secretase activity, synaptic and memory deficits, and neurodegeneration caused by Psen mutations in mouse models.

The mRNA translation initiation factor eIF4G1 controls mitochondrial oxidative phosphorylation, axonal morphogenesis, and memory.

mRNA translation initiation plays a critical role in learning and memory. The eIF4F complex, composed of the cap-binding protein eIF4E, ATP-dependent RNA helicase eIF4A, and scaffolding protein eIF4G, is a pivotal factor in the mRNA translation initiation process. eIF4G1, the major paralogue of the three eIF4G family members, is indispensable for development, but its function in learning and memory is unknown. To study the role of eIF4G1 in cognition, we used an eIF4G1 haploinsufficient (eIF4G1-1D) mouse model. The axonal arborization of eIF4G1-1D primary hippocampal neurons was significantly disrupted, and the mice displayed impairment in hippocampus-dependent learning and memory. Translatome analysis showed that the translation of mRNAs encoding proteins of the mitochondrial oxidative phosphorylation (OXPHOS) system was decreased in the eIF4G1-1D brain, and OXPHOS was decreased in eIF4G1-silenced cells. Thus, eIF4G1-mediated mRNA translation is crucial for optimal cognitive function, which is dependent on OXPHOS and neuronal morphogenesis.

The Drosophila NPY-like system protects against chronic stress-induced learning deficit by preventing the disruption of autophagic flux.

Chronic stress may induce learning and memory deficits that are associated with a depression-like state in Drosophila melanogaster. The molecular and neural mechanisms underlying the etiology of chronic stress-induced learning deficit (CSLD) remain elusive. Here, we show that the autophagy-lysosomal pathway, a conserved cellular signaling mechanism, is associated with chronic stress in Drosophila, as indicated by time-series transcriptome profiling. Our findings demonstrate that chronic stress induces the disruption of autophagic flux, and chronic disruption of autophagic flux could lead to a learning deficit. Remarkably, preventing the disruption of autophagic flux by up-regulating the basal autophagy level is sufficient to protect against CSLD. Consistent with the essential role of the dopaminergic system in modulating susceptibility to CSLD, dopamine neuronal activity is also indispensable for chronic stress to induce the disruption of autophagic flux. By screening knockout mutants, we found that neuropeptide F, the Drosophila homolog of neuropeptide Y, is necessary for normal autophagic flux and promotes resilience to CSLD. Moreover, neuropeptide F signaling during chronic stress treatment promotes resilience to CSLD by preventing the disruption of autophagic flux. Importantly, neuropeptide F receptor activity in dopamine neurons also promotes resilience to CSLD. Together, our data elucidate a mechanism by which stress-induced excessive dopaminergic activity precipitates the disruption of autophagic flux, and chronic disruption of autophagic flux leads to CSLD, while inhibitory neuropeptide F signaling to dopamine neurons promotes resilience to CSLD by preventing the disruption of autophagic flux.

Distinct Neural Plasticity Enhancing Visual Perception.

The developed human brain shows remarkable plasticity following perceptual learning, resulting in improved visual sensitivity. However, such improvements commonly require extensive stimuli exposure. Here we show that efficiently enhancing visual perception with minimal stimuli exposure recruits distinct neural mechanisms relative to standard repetition-based learning. Participants (n = 20, 12 women, 8 men) encoded a visual discrimination task, followed by brief memory reactivations of only five trials each performed on separate days, demonstrating improvements comparable with standard repetition-based learning (n = 20, 12 women, 8 men). Reactivation-induced learning engaged increased bilateral intraparietal sulcus (IPS) activity relative to repetition-based learning. Complementary evidence for differential learning processes was further provided by temporal-parietal resting functional connectivity changes, which correlated with behavioral improvements. The results suggest that efficiently enhancing visual perception with minimal stimuli exposure recruits distinct neural processes, engaging higher-order control and attentional resources while leading to similar perceptual gains. These unique brain mechanisms underlying improved perceptual learning efficiency may have important implications for daily life and in clinical conditions requiring relearning following brain damage.

Deafness causing neuroplastin missense variants fail to promote plasma membrane Ca2+-ATPase levels and Ca2+ transient regulation in brain neurons.

Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca2+) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular levels. Computational molecular dynamics show that loss of the disulfide bridge in hNppitch causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNpaudio-1 results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNpaudio-1 was confirmed by PNGaseF treatment. In comparison to hNpWT, transfection of hNppitch and hNpaudio-1 into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNppitch and hNpaudio-1 failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNppitch or hNpaudio-1 was less efficient than hNpWT to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca2+ levels after electrically evoked Ca2+ transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca2+ regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.

Loss of all three APP family members during development impairs synaptic function and plasticity, disrupts learning, and causes an autism-like phenotype.

The key role of APP for Alzheimer pathogenesis is well established. However, perinatal lethality of germline knockout mice lacking the entire APP family has so far precluded the analysis of its physiological functions for the developing and adult brain. Here, we generated conditional APP/APLP1/APLP2 triple KO (cTKO) mice lacking the APP family in excitatory forebrain neurons from embryonic day 11.5 onwards. NexCre cTKO mice showed altered brain morphology with agenesis of the corpus callosum and disrupted hippocampal lamination. Further, NexCre cTKOs revealed reduced basal synaptic transmission and drastically reduced long-term potentiation that was associated with reduced dendritic length and reduced spine density of pyramidal cells. With regard to behavior, lack of the APP family leads not only to severe impairments in a panel of tests for learning and memory, but also to an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction. Together, our study identifies essential functions of the APP family during development, for normal hippocampal function and circuits important for learning and social behavior.

Identification of differential m6A RNA methylomes and ALKBH5 as a potential prevention target in the developmental neurotoxicity induced by multiple sevoflurane exposures.

Sevoflurane, as a commonly used inhaled anesthetic for pediatric patients, has been reported that multiple sevoflurane exposures are associated with a greater risk of developing neurocognitive disorder. N6-Methyladenosine (m6A), as the most common mRNA modification in eukaryotes, has emerged as a crucial regulator of brain function in processes involving synaptic plasticity, learning and memory, and neurodevelopment. Nevertheless, the relevance of m6A RNA methylation in the multiple sevoflurane exposure-induced developmental neurotoxicity remains mostly elusive. Herein, we evaluated the genome-wide m6A RNA modification and gene expression in hippocampus of mice that received with multiple sevoflurane exposures using m6A-sequencing (m6A-seq) and RNA-sequencing (RNA-seq). We discovered 19 genes with differences in the m6A methylated modification and differential expression in the hippocampus. Among these genes, we determined that a total of nine differential expressed genes may be closely associated with the occurrence of developmental neurotoxicity induced by multiple sevoflurane exposures. We further found that the alkB homolog 5 (ALKBH5), but not methyltransferase-like 3 (METTL3) and Wilms tumor 1-associated protein (WTAP), were increased in the hippocampus of mice that received with multiple sevoflurane exposures. And the IOX1, as an inhibitor of ALKBH5, significantly improved the learning and memory defects and reduced neuronal damage in the hippocampus of mice induced by multiple sevoflurane exposures. The current study revealed the role of m6A methylated modification and m6A-related regulators in sevoflurane-induced cognitive impairment, which might provide a novel insight into identifying biomarkers and therapeutic strategies for inhaled anesthetic-induced developmental neurotoxicity.