Generation of a homozygous ABCA7-knockout human iPSC line using the CRISPR/Cas9 system.

ABCA7 encodes an ATP-binding cassette transporter, and its loss-of-function variants are associated with Alzheimer's disease. To investigate the role of ABCA7 deficiency in the pathogenesis of Alzheimer's disease, we generated a homozygous ABCA7-knockout induced pluripotent stem cell (iPSC) line using CRISPR/Cas9-mediated gene editing. This ABCA7-deficient iPSC line maintains a normal karyotype, expression of pluripotency markers, and trilineage differentiation capacity.

α2-Chimaerin is essential for neural stem cell homeostasis in mouse adult neurogenesis.

Adult hippocampal neurogenesis involves the lifelong generation of neurons. The process depends on the homeostasis of the production of neurons and maintenance of the adult neural stem cell (NSC) pool. Here, we report that α2-chimaerin, a Rho GTPase-activating protein, is essential for NSC homeostasis in adult hippocampal neurogenesis. Conditional deletion of α2-chimaerin in adult NSCs resulted in the premature differentiation of NSCs into intermediate progenitor cells (IPCs), which ultimately depleted the NSC pool and impaired neuron generation. Single-cell RNA sequencing and pseudotime analyses revealed that α2-chimaerin-conditional knockout (α2-CKO) mice lacked a unique NSC subpopulation, termed Klotho-expressing NSCs, during the transition of NSCs to IPCs. Furthermore, α2-CKO led to defects in hippocampal synaptic plasticity and anxiety/depression-like behaviors in mice. Our findings collectively demonstrate that α2-chimaerin plays an essential role in adult hippocampal NSC homeostasis to maintain proper brain function.

Locally coordinated synaptic plasticity of visual cortex neurons in vivo.

Plasticity of cortical responses in vivo involves activity-dependent changes at synapses, but the manner in which different forms of synaptic plasticity act together to create functional changes in neurons remains unknown. We found that spike timing-induced receptive field plasticity of visual cortex neurons in mice is anchored by increases in the synaptic strength of identified spines. This is accompanied by a decrease in the strength of adjacent spines on a slower time scale. The locally coordinated potentiation and depression of spines involves prominent AMPA receptor redistribution via targeted expression of the immediate early gene product Arc. Hebbian strengthening of activated synapses and heterosynaptic weakening of adjacent synapses thus cooperatively orchestrate cell-wide plasticity of functional neuronal responses.

Rett syndrome: insights into genetic, molecular and circuit mechanisms.

Rett syndrome (RTT) is a severe neurological disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). Almost two decades of research into RTT have greatly advanced our understanding of the function and regulation of the multifunctional protein MeCP2. Here, we review recent advances in understanding how loss of MeCP2 impacts different stages of brain development, discuss recent findings demonstrating the molecular role of MeCP2 as a transcriptional repressor, assess primary and secondary effects of MeCP2 loss and examine how loss of MeCP2 can result in an imbalance of neuronal excitation and inhibition at the circuit level along with dysregulation of activity-dependent mechanisms. These factors present challenges to the search for mechanism-based therapeutics for RTT and suggest specific approaches that may be more effective than others.

Major Vault Protein, a Candidate Gene in 16p11.2 Microdeletion Syndrome, Is Required for the Homeostatic Regulation of Visual Cortical Plasticity.

Microdeletion of a region in chromosome 16p11.2 increases susceptibility to autism. Although this region contains exons of 29 genes, disrupting only a small segment of the region, which spans five genes, is sufficient to cause autistic traits. One candidate gene in this critical segment is MVP, which encodes for the major vault protein (MVP) that has been implicated in regulation of cellular transport mechanisms. MVP expression levels in MVP+/- mice closely phenocopy those of 16p11.2 mutant mice, suggesting that MVP+/- mice may serve as a model of MVP function in 16p11.2 microdeletion. Here we show that MVP regulates the homeostatic component of ocular dominance (OD) plasticity in primary visual cortex. MVP+/- mice of both sexes show impairment in strengthening of open-eye responses after several days of monocular deprivation (MD), whereas closed-eye responses are weakened as normal, resulting in reduced overall OD plasticity. The frequency of miniature EPSCs (mEPSCs) in pyramidal neurons is decreased in MVP+/- mice after extended MD, suggesting a reduction of functional synapses. Correspondingly, upregulation of surface GluA1 AMPA receptors is reduced in MVP+/- mice after extended MD, and is accompanied by altered expression of STAT1 and phosphorylated ERK, which have been previously implicated in OD plasticity. Normalization of STAT1 levels by introducing STAT1 shRNA rescues surface GluA1 and open-eye responses, implicating STAT1 as a downstream effector of MVP. These findings demonstrate a specific role for MVP as a key molecule influencing the homeostatic component of activity-dependent synaptic plasticity, and potentially the corresponding phenotypes of 16p11.2 microdeletion syndrome.SIGNIFICANCE STATEMENT Major vault protein (MVP), a candidate gene in 16p11.2 microdeletion syndrome, has been implicated in the regulation of several cellular processes including transport mechanisms and scaffold signaling. However, its role in brain function and plasticity remains unknown. In this study, we identified MVP as an important regulator of the homeostatic component of experience-dependent plasticity, via regulation of STAT1 and ERK signaling. This study helps reveal a new mechanism for an autism-related gene in brain function, and suggests a broader role for neuro-immune interactions in circuit level plasticity. Importantly, our findings might explain specific components of the pathophysiology of 16p11.2 microdeletion syndrome.

Cdk5-mediated phosphorylation of RapGEF2 controls neuronal migration in the developing cerebral cortex.

During cerebral cortex development, pyramidal neurons migrate through the intermediate zone and integrate into the cortical plate. These neurons undergo the multipolar-bipolar transition to initiate radial migration. While perturbation of this polarity acquisition leads to cortical malformations, how this process is initiated and regulated is largely unknown. Here we report that the specific upregulation of the Rap1 guanine nucleotide exchange factor, RapGEF2, in migrating neurons corresponds to the timing of this polarity transition. In utero electroporation and live-imaging studies reveal that RapGEF2 acts on the multipolar-bipolar transition during neuronal migration via a Rap1/N-cadherin pathway. Importantly, activation of RapGEF2 is controlled via phosphorylation by a serine/threonine kinase Cdk5, whose activity is largely restricted to the radial migration zone. Thus, the specific expression and Cdk5-dependent phosphorylation of RapGEF2 during multipolar-bipolar transition within the intermediate zone are essential for proper neuronal migration and wiring of the cerebral cortex.

Cdk5-dependent Mst3 phosphorylation and activity regulate neuronal migration through RhoA inhibition.

The radial migration of newborn neurons is critical for the lamination of the cerebral cortex. Proper neuronal migration requires precise and rapid reorganization of the actin and microtubule cytoskeleton. However, the underlying signaling mechanisms controlling cytoskeletal reorganization are not well understood. Here, we show that Mst3, a serine/threonine kinase highly expressed in the developing mouse brain, is essential for radial neuronal migration and final neuronal positioning in the developing mouse neocortex. Mst3 silencing by in utero electroporation perturbed the multipolar-to-bipolar transition of migrating neurons and significantly retards radial migration. Although the kinase activity of Mst3 is essential for its functions in neuronal morphogenesis and migration, it is regulated via its phosphorylation at Ser79 by a serine/threonine kinase, cyclin-dependent kinase 5 (Cdk5). Our results show that Mst3 regulates neuronal migration through modulating the activity of RhoA, a Rho-GTPase critical for actin cytoskeletal reorganization. Mst3 phosphorylates RhoA at Ser26, thereby negatively regulating the GTPase activity of RhoA. Importantly, RhoA knockdown successfully rescues neuronal migration defect in Mst3-knockdown cortices. Our findings collectively suggest that Cdk5-Mst3 signaling regulates neuronal migration via RhoA-dependent actin dynamics.

CRMP2: functional roles in neural development and therapeutic potential in neurological diseases.

Cytoskeletal restructuring is essential for nearly all cellular processes in the developing brain. After cell fate determination, newborn cortical neurons must migrate to their final positions while establishing proper axon-dendrite polarity. Significant progress has recently been made towards understanding the cellular and molecular mechanisms underlying neuronal polarization in vivo. Collapsin response mediator protein 2 (CRMP2) has long been identified as a microtubule-binding protein that regulates neuronal polarity in vitro. Recent studies provide new insights into the roles of CRMP2 in neuronal migration and subsequent neuronal differentiation. Both the expression and activity of CRMP2 are tightly regulated during cortex development. CRMP2 is suggested to be important in the multipolar-bipolar transition in radial migration. The increasing number of known interaction partners indicates that CRMP2 has functions beyond cytoskeletal regulation, including axonal transport, vesicle trafficking, and neurotransmitter release. This review discusses the current knowledge about CRMP2 in the context of neuronal development and highlights a recent emerging theme regarding its potential therapeutic applications.

The bacteriostatic protein lipocalin 2 is induced in the central nervous system of mice with west Nile virus encephalitis.

Lipocalin 2 (Lcn2) is a bacteriostatic factor produced during the innate immune response to bacterial infection. Whether Lcn2 has a function in viral infection is unknown. We investigated the regulation and function of Lcn2 in the central nervous system (CNS) of mice during West Nile virus (WNV) encephalitis. Lcn2 mRNA and protein were induced in the brain by day 5, and this induction increased further by day 7 postinfection but was delayed compared with the induction of the toll-like receptor 3 (TLR3) gene, retinoic acid-inducible gene 1 (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) gene. The Lcn2 mRNA and protein were both found at high levels in the choroid plexus, vascular endothelium, macrophage/microglia, and astrocytes. However, some neuronal subsets contained Lcn2 protein but no detectable mRNA. In Lcn2 knockout (KO) mice, with the exception of CXC motif chemokine 5 (CXCL5), which was significantly more downregulated than in wild-type (WT) mice, expression levels of a number of other host response genes were similar in the two genotypes. The brain from Lcn2 and WT mice with WNV encephalitis contained similar numbers of infiltrating macrophages, granulocytes, and T cells. Lcn2 KO and WT mice had no significant difference in tissue viral loads or survival after infection with different doses of WNV. We conclude that Lcn2 gene expression is induced to high levels in a time-dependent fashion in a variety of cells and regions of the CNS of mice with WNV encephalitis. The function of Lcn2 in the host response to WNV infection remains largely unknown, but our data indicate that it is dispensable as an antiviral or immunoregulatory factor in WNV encephalitis.

Ankyrin repeat-rich membrane spanning protein (kidins220) is required for neurotrophin and ephrin receptor-dependent dendrite development.

Dendrites are the primary sites on neurons for receiving and integrating inputs from their presynaptic partners. Defects in dendrite development perturb the formation of neural circuitry and impair information processing in the brain. Extracellular cues are important for shaping the dendritic morphogenesis, but the underlying molecular mechanisms are not well understood. In this study, we examined the role of ARMS (ankyrin repeat-rich membrane spanning protein), also known as Kidins220 (kinase D-interacting substrate of 220 kDa), previously identified as a downstream target of neurotrophin and ephrin receptors, in dendrite development. We report here that knockdown of ARMS/Kidins220 by in utero electroporation impairs dendritic branching in mouse cerebral cortex, and silencing of ARMS/Kidins220 in primary rat hippocampal neurons results in a significant decrease in the length, number, and complexity of the dendritic arbors. Overexpression of cell surface receptor tyrosine kinases, including TrkB and EphB2, in ARMS/Kidins220-deficient neurons can partially rescue the defective dendritic phenotype. More importantly, we show that PI3K (phosphoinositide-3-kinase)- and Akt-mediated signaling pathway is crucial for ARMS/Kidins220-dependent dendrite development. Furthermore, loss of ARMS/Kidins220 significantly reduced the clustering of EphB2 receptor signaling complex in neurons. Our results collectively suggest that ARMS/Kidins220 is a key player in organizing the signaling complex to transduce the extracellular stimuli to cellular responses during dendrite development.

α2-chimaerin controls neuronal migration and functioning of the cerebral cortex through CRMP-2.

Disrupted cortical neuronal migration is associated with epileptic seizures and developmental delay. However, the molecular mechanism by which disruptions of early cortical development result in neurological symptoms is poorly understood. Here we report α2-chimaerin as a key regulator of cortical neuronal migration and function. In utero suppression of α2-chimaerin arrested neuronal migration at the multipolar stage, leading to accumulation of ectopic neurons in the subcortical region. Mice with such migration defects showed an imbalance between excitation and inhibition in local cortical circuitry and greater susceptibility to convulsant-induced seizures. We further show that α2-chimaerin regulates bipolar transition and neuronal migration through modulating the activity of CRMP-2, a microtubule-associated protein. These findings establish a new α2-chimaerin-dependent mechanism underlying neuronal migration and proper functioning of the cerebral cortex and provide insights into the pathogenesis of seizure-related neurodevelopmental disorders.

Lipocalin 2 in the central nervous system host response to systemic lipopolysaccharide administration.

BACKGROUND: Lipocalin 2 (Lcn2) is a bacteriostatic factor that may also modulate cellular function, however, little is known concerning the expression or role of Lcn2 in CNS inflammation. Therefore, here we investigated the regulation and possible function of Lcn2 in the CNS following peripheral lipopolysaccharide (LPS) injection in mice. METHODS: A murine model for systemic endotoxemia was used in this study. Wild type or Lcn2 KO mice (both genotypes C57BL/6 strain) were given either a single or dual, staggered intraperitoneal injections of purified E. coli LPS or vehicle alone. The brain was examined for the expression and location of Lcn2 mRNA and protein and various markers for neuroinflammation were analyzed. RESULTS: Although undetectable under physiological conditions, both Lcn2 mRNA and protein were induced to high levels in the brain after LPS injection. By contrast, RNA corresponding to the putative Lcn2 (termed 24p3R) receptor was present at high levels in the normal brain and remained unaltered by LPS injection. Differences between Lcn2 and 24p3R mRNA expression were found at the anatomic and cellular level. Endothelial cells, microglia and the choroid plexus but not neurons were identified as the main cellular sources for Lcn2 mRNA in the CNS. By contrast, 24p3R mRNA was detected in neurons and the choroid plexus only. Lcn2 protein was found to have a similar cellular localization as the corresponding RNA transcripts with the exception that subsets of neurons were also strongly positive. Various inflammatory, glial, and iron handling markers were analyzed and found to have similar alterations between WT and Lcn2 KO animals. CONCLUSIONS: 1) Lcn2 production is strongly induced in the CNS by systemic LPS injection, 2) in addition to Lcn2 production at key gateways of bacterial entry to the CNS, neurons may be a target for the actions of Lcn2, which is apparently taken up by these cells, and 3) the cellular functions of Lcn2 in the CNS remain enigmatic.

Cdk5-mediated phosphorylation of Axin directs axon formation during cerebral cortex development.

Axon formation is critical for the establishment of connections between neurons, which is a prerequisite for the development of neural circuitry. Kinases such as cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase-3ß (GSK-3ß), have been implicated to regulate axon outgrowth. Nonetheless, the in vivo roles of these kinases in axon development and the underlying signaling mechanisms remain essentially unknown. We report here that Cdk5 is important for axon formation in mouse cerebral cortex through regulating the functions of axis inhibitor (Axin), a scaffold protein of the canonical Wnt pathway. Knockdown of Axin in utero abolishes the formation and projection of axons. Importantly, Axin is phosphorylated by Cdk5, and this phosphorylation facilitates the interaction of Axin with GSK-3ß, resulting in inhibition of GSK-3ß activity and dephosphorylation of its substrate collapsin response mediator protein-2 (CRMP-2), a microtubule-associated protein. Specifically, both phosphorylation of Axin and its interaction with GSK-3ß are critically required for axon formation in mouse cortex development. Together, our findings reveal a new regulatory mechanism of axon formation through Cdk5-dependent phosphorylation of Axin.

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm.

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.