Synaptic ensembles between raphe and D1R-containing accumbens shell neurons underlie postisolation sociability in males.

Social animals expend considerable energy to maintain social bonds throughout their life. Male and female mice show sexually dimorphic behaviors, yet the underlying neural mechanisms of sociability and their dysregulation during social disconnection remain unknown. Dopaminergic neurons in dorsal raphe nucleus (DRNTH) is known to contribute to a loneliness-like state and modulate sociability. We identified that activated subpopulations in DRNTH and nucleus accumbens shell (NAcsh) during 24 hours of social isolation underlie the increase in isolation-induced sociability in male but not in female mice. This effect was reversed by chemogenetically and optogenetically inhibiting the DRNTH-NAcsh circuit. Moreover, synaptic connectivity among the activated neuronal ensembles in this circuit was increased, primarily in D1 receptor-expressing neurons in NAcsh. The increase in synaptic density functionally correlated with elevated dopamine release into NAcsh. Overall, specific synaptic ensembles in DRNTH-NAcsh mediate sex differences in isolation-induced sociability, indicating that sex-dependent circuit dynamics underlie the expression of sexually dimorphic behaviors.

Interactome analysis illustrates diverse gene regulatory processes associated with LIN28A in human iPS cell-derived neural progenitor cells.

A single protein can be multifaceted depending on the cellular contexts and interacting molecules. LIN28A is an RNA-binding protein that governs developmental timing, cellular proliferation, differentiation, stem cell pluripotency, and metabolism. In addition to its best-known roles in microRNA biogenesis, diverse molecular roles have been recognized. In the nervous system, LIN28A is known to play critical roles in proliferation and differentiation of neural progenitor cells (NPCs). We profiled the endogenous LIN28A-interacting proteins in NPCs differentiated from human induced pluripotent stem (iPS) cells using immunoprecipitation and liquid chromatography-tandem mass spectrometry. We identified over 500 LIN28A-interacting proteins, including 156 RNA-independent interactors. Functions of these proteins span a wide range of gene regulatory processes. Prompted by the interactome data, we revealed that LIN28A may impact the subcellular distribution of its interactors and stress granule formation upon oxidative stress. Overall, our analysis opens multiple avenues for elaborating molecular roles and characteristics of LIN28A.

Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.

Reciprocal deletion and duplication of the 16p11.2 region is the most common copy number variation (CNV) associated with autism spectrum disorders. We generated cortical organoids from skin fibroblasts of patients with 16p11.2 CNV to investigate impacted neurodevelopmental processes. We show that organoid size recapitulates macrocephaly and microcephaly phenotypes observed in the patients with 16p11.2 deletions and duplications. The CNV dosage affects neuronal maturation, proliferation, and synapse number, in addition to its effect on organoid size. We demonstrate that 16p11.2 CNV alters the ratio of neurons to neural progenitors in organoids during early neurogenesis, with a significant excess of neurons and depletion of neural progenitors observed in deletions. Transcriptomic and proteomic profiling revealed multiple pathways dysregulated by the 16p11.2 CNV, including neuron migration, actin cytoskeleton, ion channel activity, synaptic-related functions, and Wnt signaling. The level of the active form of small GTPase RhoA was increased in both, deletions and duplications. Inhibition of RhoA activity rescued migration deficits, but not neurite outgrowth. This study provides insights into potential neurobiological mechanisms behind the 16p11.2 CNV during neocortical development.

Altered network and rescue of human neurons derived from individuals with early-onset genetic epilepsy.

Early-onset epileptic encephalopathies are severe disorders often associated with specific genetic mutations. In this context, the CDKL5 deficiency disorder (CDD) is a neurodevelopmental condition characterized by early-onset seizures, intellectual delay, and motor dysfunction. Although crucial for proper brain development, the precise targets of CDKL5 and its relation to patients' symptoms are still unknown. Here, induced pluripotent stem cells derived from individuals deficient in CDKL5 protein were used to generate neural cells. Proteomic and phosphoproteomic approaches revealed disruption of several pathways, including microtubule-based processes and cytoskeleton organization. While CDD-derived neural progenitor cells have proliferation defects, neurons showed morphological alterations and compromised glutamatergic synaptogenesis. Moreover, the electrical activity of CDD cortical neurons revealed hyperexcitability during development, leading to an overly synchronized network. Many parameters of this hyperactive network were rescued by lead compounds selected from a human high-throughput drug screening platform. Our results enlighten cellular, molecular, and neural network mechanisms of genetic epilepsy that could ultimately promote novel therapeutic opportunities for patients.

Autism-linked Cullin3 germline haploinsufficiency impacts cytoskeletal dynamics and cortical neurogenesis through RhoA signaling.

E3-ubiquitin ligase Cullin3 (Cul3) is a high confidence risk gene for autism spectrum disorder (ASD) and developmental delay (DD). To investigate how Cul3 mutations impact brain development, we generated a haploinsufficient Cul3 mouse model using CRISPR/Cas9 genome engineering. Cul3 mutant mice exhibited social and cognitive deficits and hyperactive behavior. Brain MRI found decreased volume of cortical regions and changes in many other brain regions of Cul3 mutant mice starting from early postnatal development. Spatiotemporal transcriptomic and proteomic profiling of embryonic, early postnatal and adult brain implicated neurogenesis and cytoskeletal defects as key drivers of Cul3 functional impact. Specifically, dendritic growth, filamentous actin puncta, and spontaneous network activity were reduced in Cul3 mutant mice. Inhibition of small GTPase RhoA, a molecular substrate of Cul3 ligase, rescued dendrite length and network activity phenotypes. Our study identified defects in neuronal cytoskeleton and Rho signaling as the primary targets of Cul3 mutation during brain development.

Conditional knock out of transcription factor CTCF in excitatory neurons induces cognitive deficiency.

CCCTC-binding factor (CTCF) is a transcription factor that is involved in organizing chromatin structure. A reduction of CTCF expression is known to develop distinct clinical features. Furthermore, conditional knock out (cKO) study revealed reactive gliosis of astrocytes and microglia followed by age-dependent cell death in the excitatory neurons of CTCF cKO mice. To assess the cognitive ability in CTCF cKO mice of over 20 weeks of age, we examined pairwise discrimination (PD), PD reversal learning (PDr), and different paired-associate learning (dPAL) tasks using a touch screen apparatus. We found cognitive impairment in dPAL touch screen tests, suggesting that prolonged Ctcf gene deficiency results in cognitive deficits.

Loss of the neuronal genome organizer and transcription factor CTCF induces neuronal death and reactive gliosis in the anterior cingulate cortex.

CCCTC-binding factor (CTCF) is a genome organizer that regulates gene expression through transcription and chromatin structure regulation. CTCF also plays an important role during the developmental and adult stages. Cell-specific CTCF deletion studies have shown that a reduction in CTCF expression leads to the development of distinct clinical features and cognitive disorders. Therefore, we knocked out Ctcf (CTCF cKO) in the excitatory neurons of the forebrain in a Camk2a-Cre mouse strain to examine the role of CTCF in cell death and gliosis in the cortex. CTCF cKO mice were viable, but they demonstrated an age-dependent increase in reactive gliosis of astrocytes and microglia in the anterior cingulate cortex (ACC) from 16 weeks of age prior to neuronal loss observed at over 20 weeks of age. Consistent with these data, qRT-PCR analysis of the CTCF cKO ACC revealed changes in the expression of inflammation-related genes (Hspa1a, Prokr2 and Itga8) linked to gliosis and neuronal death. Our results suggest that prolonged Ctcf gene deficiency in excitatory neurons results in neuronal cell death and gliosis, possibly through functional changes in inflammation-related genes.

Impact of the Identification Strategy on the Reproducibility of the DDA and DIA Results.

Data-independent acquisition (DIA) is a promising technique for the proteomic analysis of complex protein samples. A number of studies have claimed that DIA experiments are more reproducible than data-dependent acquisition (DDA), but these claims are unsubstantiated since different data analysis methods are used in the two methods. Data analysis in most DIA workflows depends on spectral library searches, whereas DDA typically employs sequence database searches. In this study, we examined the reproducibility of the DIA and DDA results using both sequence database and spectral library search. The comparison was first performed using a cell lysate and then extended to an interactome study. Protein overlap among the technical replicates in both DDA and DIA experiments was 30% higher with library-based identifications than with sequence database identifications. The reproducibility of quantification was also improved with library search compared to database search, with the mean of the coefficient of variation decreasing more than 30% and a reduction in the number of missing values of more than 35%. Our results show that regardless of the acquisition method, higher identification and quantification reproducibility is observed when library search was used.

Identification of a novel Shank2 transcriptional variant in Shank2 knockout mouse model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders that are highly heterogeneous in clinical symptoms as well as etiologies. Mutations in SHANK2 are associated with ASD and accordingly, Shank2 knockout mouse shows ASD-like behavioral phenotypes, including social deficits. Intriguingly, two lines of Shank2 knockout (KO) mouse generated by deleting different exons (exon 6-7 or exon 7) showed distinct cellular phenotypes. Previously, we compared gene expressions between Shank2 KOs lacking exon 6-7 (e6-7 KO) and KOs lacking exon 7 (e7 KO) by performing RNA-seq. In this study, we expanded transcriptomic analyses to identify novel transcriptional variants in the KO mice. We found prominent expression of a novel exon (exon 4' or e4') between the existing exons 4 and 5 in the Shank2 e6-7 KO model. Expression of the transcriptional variant harboring this novel exon was confirmed by RT-PCR and western blotting. These findings suggest that the novel variant may function as a modifier gene, which contributes to the differences between the two Shank2 mutant lines. Furthermore, our result further represents an example of genetic compensation that may lead to phenotypic heterogeneity among ASD patients with mutations in the same gene.

Structural Analysis of Hippocampal Kinase Signal Transduction.

Kinases are a major clinical target for human diseases. Identifying the proteins that interact with kinases in vivo will provide information on unreported substrates and will potentially lead to more specific methods for therapeutic kinase regulation. Here, endogenous immunoprecipitations of evolutionally distinct kinases (i.e., Akt, ERK2, and CAMK2) from rodent hippocampi were analyzed by mass spectrometry to generate three highly confident kinase protein-protein interaction networks. Proteins of similar function were identified in the networks, suggesting a universal model for kinase signaling complexes. Protein interactions were observed between kinases with reported symbiotic relationships. The kinase networks were significantly enriched in genes associated with specific neurodevelopmental disorders providing novel structural connections between these disease-associated genes. To demonstrate a functional relationship between the kinases and the network, pharmacological manipulation of Akt in hippocampal slices was shown to regulate the activity of potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel(HCN1), which was identified in the Akt network. Overall, the kinase protein-protein interaction networks provide molecular insight of the spatial complexity of in vivo kinase signal transduction which is required to achieve the therapeutic potential of kinase manipulation in the brain.

Remote Memory and Cortical Synaptic Plasticity Require Neuronal CCCTC-Binding Factor (CTCF).

The molecular mechanism of long-term memory has been extensively studied in the context of the hippocampus-dependent recent memory examined within several days. However, months-old remote memory maintained in the cortex for long-term has not been investigated much at the molecular level yet. Various epigenetic mechanisms are known to be important for long-term memory, but how the 3D chromatin architecture and its regulator molecules contribute to neuronal plasticity and systems consolidation is still largely unknown. CCCTC-binding factor (CTCF) is an 11-zinc finger protein well known for its role as a genome architecture molecule. Male conditional knock-out mice in which CTCF is lost in excitatory neurons during adulthood showed normal recent memory in the contextual fear conditioning and spatial water maze tasks. However, they showed remarkable impairments in remote memory in both tasks. Underlying the remote memory-specific phenotypes, we observed that female CTCF conditional knock-out mice exhibit disrupted cortical LTP, but not hippocampal LTP. Similarly, we observed that CTCF deletion in inhibitory neurons caused partial impairment of remote memory. Through RNA sequencing, we observed that CTCF knockdown in cortical neuron culture caused altered expression of genes that are highly involved in cell adhesion, synaptic plasticity, and memory. These results suggest that remote memory storage in the cortex requires CTCF-mediated gene regulation in neurons, whereas recent memory formation in the hippocampus does not.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a well-known 3D genome architectural protein that regulates gene expression. Here, we use two different CTCF conditional knock-out mouse lines and reveal, for the first time, that CTCF is critically involved in the regulation of remote memory. We also show that CTCF is necessary for appropriate expression of genes, many of which we found to be involved in the learning- and memory-related processes. Our study provides behavioral and physiological evidence for the involvement of CTCF-mediated gene regulation in the remote long-term memory and elucidates our understanding of systems consolidation mechanisms.

Increased PKMζ activity impedes lateral movement of GluA2-containing AMPA receptors.

Protein kinase M zeta (PKMζ), a constitutively active, atypical protein kinase C isoform, maintains a high level of expression in the brain after the induction of learning and long-term potentiation (LTP). Further, its overexpression enhances long-term memory and LTP. Thus, multiple lines of evidence suggest a significant role for persistently elevated PKMζ levels in long-term memory. The molecular mechanisms of how synaptic properties are regulated by the increase in PKMζ, however, are still largely unknown. The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) mediates most of the fast glutamatergic synaptic transmission in the brain and is known to be critical for the expression of synaptic plasticity and memory. Importance of AMPAR trafficking has been implicated in PKMζ-mediated cellular processes, but the detailed mechanisms, particularly in terms of regulation of AMPAR lateral movement, are not well understood. In the current study, using a single-molecule live imaging technique, we report that the overexpression of PKMζ in hippocampal neurons immobilized GluA2-containing AMPARs, highlighting a potential novel mechanism by which PKMζ may regulate memory and synaptic plasticity.

Enhancing inhibitory synaptic function reverses spatial memory deficits in Shank2 mutant mice.

Autism spectrum disorders (ASDs) are a group of developmental disorders that cause variable and heterogeneous phenotypes across three behavioral domains such as atypical social behavior, disrupted communications, and highly restricted and repetitive behaviors. In addition to these core symptoms, other neurological abnormalities are associated with ASD, including intellectual disability (ID). However, the molecular etiology underlying these behavioral heterogeneities in ASD is unclear. Mutations in SHANK2 genes are associated with ASD and ID. Interestingly, two lines of Shank2 knockout mice (e6-7 KO and e7 KO) showed shared and distinct phenotypes. Here, we found that the expression levels of Gabra2, as well as of GABA receptor-mediated inhibitory neurotransmission, are reduced in Shank2 e6-7, but not in e7 KO mice compared with their own wild type littermates. Furthermore, treatment of Shank2 e6-7 KO mice with an allosteric modulator for the GABAA receptor reverses spatial memory deficits, indicating that reduced inhibitory neurotransmission may cause memory deficits in Shank2 e6-7 KO mice. This article is part of the Special Issue entitled 'Ionotropic glutamate receptors'.

Response to Comment on "Multiple repressive mechanisms in the hippocampus during memory formation".

Mathew et al. propose that many candidate genes identified in our study may reflect the events in the choroid plexus (ChP) potentially included in hippocampal samples. We reanalyze our data and find that the ChP inclusion is unlikely to affect our major conclusions regarding the basal suppression of translational machinery or the early translational repression (at 5 to 10 minutes). As Mathew et al. examined for a subset of genes at 4 hours, we agree that the late suppression may partly reflect the events in the ChP. Although the precise contribution of anatomical sources remains to be clarified, our behavioral analyses indicate that the late-phase suppression of these genes may contribute to memory formation.

The Brain-Enriched MicroRNA miR-9-3p Regulates Synaptic Plasticity and Memory.

UNLABELLED: MicroRNAs (miRNAs) are small, noncoding RNAs that posttranscriptionally regulate gene expression in many tissues. Although a number of brain-enriched miRNAs have been identified, only a few specific miRNAs have been revealed as critical regulators of synaptic plasticity, learning, and memory. miR-9-5p/3p are brain-enriched miRNAs known to regulate development and their changes have been implicated in several neurological disorders, yet their role in mature neurons in mice is largely unknown. Here, we report that inhibition of miR-9-3p, but not miR-9-5p, impaired hippocampal long-term potentiation (LTP) without affecting basal synaptic transmission. Moreover, inhibition of miR-9-3p in the hippocampus resulted in learning and memory deficits. Furthermore, miR-9-3p inhibition increased the expression of the LTP-related genes Dmd and SAP97, the expression levels of which are negatively correlated with LTP. These results suggest that miR-9-3p-mediated gene regulation plays important roles in synaptic plasticity and hippocampus-dependent memory. SIGNIFICANCE STATEMENT: Despite the abundant expression of the brain-specific microRNA miR-9-5p/3p in both proliferating and postmitotic neurons, most functional studies have focused on their role in neuronal development. Here, we examined the role of miR-9-5p/3p in adult brain and found that miR-9-3p, but not miR-9-5p, has a critical role in hippocampal synaptic plasticity and memory. Moreover, we identified in vivo binding targets of miR-9-3p that are involved in the regulation of long-term potentiation. Our study provides the very first evidence for the critical role of miR-9-3p in synaptic plasticity and memory in the adult mouse.

Gene repressive mechanisms in the mouse brain involved in memory formation.

Gene regulation in the brain is essential for long-term plasticity and memory formation. Despite this established notion, the quantitative translational map in the brain during memory formation has not been reported. To systematically probe the changes in protein synthesis during memory formation, our recent study exploited ribosome profiling using the mouse hippocampal tissues at multiple time points after a learning event. Analysis of the resulting database revealed novel types of gene regulation after learning. First, the translation of a group of genes was rapidly suppressed without change in mRNA levels. At later time points, the expression of another group of genes was downregulated through reduction in mRNA levels. This reduction was predicted to be downstream of inhibition of ESR1 (Estrogen Receptor 1) signaling. Overexpressing Nrsn1, one of the genes whose translation was suppressed, or activating ESR1 by injecting an agonist interfered with memory formation, suggesting the functional importance of these findings. Moreover, the translation of genes encoding the translational machineries was found to be suppressed, among other genes in the mouse hippocampus. Together, this unbiased approach has revealed previously unidentified characteristics of gene regulation in the brain and highlighted the importance of repressive controls. [BMB Reports 2016; 49(4): 199-200].

A transducible nuclear/nucleolar protein, mLLP, regulates neuronal morphogenesis and synaptic transmission.

Cell-permeable proteins are emerging as unconventional regulators of signal transduction and providing a potential for therapeutic applications. However, only a few of them are identified and studied in detail. We identify a novel cell-permeable protein, mouse LLP homolog (mLLP), and uncover its roles in regulating neural development. We found that mLLP is strongly expressed in developing nervous system and that mLLP knockdown or overexpression during maturation of cultured neurons affected the neuronal growth and synaptic transmission. Interestingly, extracellular addition of mLLP protein enhanced dendritic arborization, demonstrating the non-cell-autonomous effect of mLLP. Moreover, mLLP interacts with CCCTC-binding factor (CTCF) as well as transcriptional machineries and modulates gene expression involved in neuronal growth. Together, these results illustrate the characteristics and roles of previously unknown cell-permeable protein mLLP in modulating neural development.

Multiple repressive mechanisms in the hippocampus during memory formation.

Memory stabilization after learning requires translational and transcriptional regulations in the brain, yet the temporal molecular changes that occur after learning have not been explored at the genomic scale. We used ribosome profiling and RNA sequencing to quantify the translational status and transcript levels in the mouse hippocampus after contextual fear conditioning. We revealed three types of repressive regulations: translational suppression of ribosomal protein-coding genes in the hippocampus, learning-induced early translational repression of specific genes, and late persistent suppression of a subset of genes via inhibition of estrogen receptor 1 (ESR1/ERα) signaling. In behavioral analyses, overexpressing Nrsn1, one of the newly identified genes undergoing rapid translational repression, or activating ESR1 in the hippocampus impaired memory formation. Collectively, this study unveils the yet-unappreciated importance of gene repression mechanisms for memory formation.