MeCP2-regulated miRNAs control early human neurogenesis through differential effects on ERK and AKT signaling.

Rett syndrome (RTT) is an X-linked, neurodevelopmental disorder caused primarily by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, which encodes a multifunctional epigenetic regulator with known links to a wide spectrum of neuropsychiatric disorders. Although postnatal functions of MeCP2 have been thoroughly investigated, its role in prenatal brain development remains poorly understood. Given the well-established importance of microRNAs (miRNAs) in neurogenesis, we employed isogenic human RTT patient-derived induced pluripotent stem cell (iPSC) and MeCP2 short hairpin RNA knockdown approaches to identify novel MeCP2-regulated miRNAs enriched during early human neuronal development. Focusing on the most dysregulated miRNAs, we found miR-199 and miR-214 to be increased during early brain development and to differentially regulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase and protein kinase B (PKB/AKT) signaling. In parallel, we characterized the effects on human neurogenesis and neuronal differentiation brought about by MeCP2 deficiency using both monolayer and three-dimensional (cerebral organoid) patient-derived and MeCP2-deficient neuronal culture models. Inhibiting miR-199 or miR-214 expression in iPSC-derived neural progenitors deficient in MeCP2 restored AKT and ERK activation, respectively, and ameliorated the observed alterations in neuronal differentiation. Moreover, overexpression of miR-199 or miR-214 in the wild-type mouse embryonic brains was sufficient to disturb neurogenesis and neuronal migration in a similar manner to Mecp2 knockdown. Taken together, our data support a novel miRNA-mediated pathway downstream of MeCP2 that influences neurogenesis via interactions with central molecular hubs linked to autism spectrum disorders.

Patient-specific models of microglia-mediated engulfment of synapses and neural progenitors.

Engulfment of synapses and neural progenitor cells (NPCs) by microglia is critical for the development and maintenance of proper brain circuitry, and has been implicated in neurodevelopmental as well as neurodegenerative disease etiology. We have developed and validated models of these mechanisms by reprogramming microglia-like cells from peripheral blood mononuclear cells, and combining them with NPCs and neurons derived from induced pluripotent stem cells to create patient-specific cellular models of complement-dependent synaptic pruning and elimination of NPCs. The resulting microglia-like cells express appropriate markers and function as primary human microglia, while patient-matched macrophages differ markedly. As a demonstration of disease-relevant application, we studied the role of C4, recently implicated in schizophrenia, in engulfment of synaptic structures by human microglia. The ability to create complete patient-specific cellular models of critical microglial functions utilizing samples taken during a single clinical visit will extend the ability to model central nervous system disease while facilitating high-throughput screening.

Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities.

Bipolar disorder (BD) is a common neuropsychiatric disorder characterized by chronic recurrent episodes of depression and mania. Despite evidence for high heritability of BD, little is known about its underlying pathophysiology. To develop new tools for investigating the molecular and cellular basis of BD, we applied a family-based paradigm to derive and characterize a set of 12 induced pluripotent stem cell (iPSC) lines from a quartet consisting of two BD-affected brothers and their two unaffected parents. Initially, no significant phenotypic differences were observed between iPSCs derived from the different family members. However, upon directed neural differentiation, we observed that CXCR4 (CXC chemokine receptor-4) expressing central nervous system (CNS) neural progenitor cells (NPCs) from both BD patients compared with their unaffected parents exhibited multiple phenotypic differences at the level of neurogenesis and expression of genes critical for neuroplasticity, including WNT pathway components and ion channel subunits. Treatment of the CXCR4(+) NPCs with a pharmacological inhibitor of glycogen synthase kinase 3, a known regulator of WNT signaling, was found to rescue a progenitor proliferation deficit in the BD patient NPCs. Taken together, these studies provide new cellular tools for dissecting the pathophysiology of BD and evidence for dysregulation of key pathways involved in neurodevelopment and neuroplasticity. Future generation of additional iPSCs following a family-based paradigm for modeling complex neuropsychiatric disorders in conjunction with in-depth phenotyping holds promise for providing insights into the pathophysiological substrates of BD and is likely to inform the development of targeted therapeutics for its treatment and ideally prevention.

Dysregulation of miR-34a links neuronal development to genetic risk factors for bipolar disorder.

Bipolar disorder (BD) is a heritable neuropsychiatric disorder with largely unknown pathogenesis. Given their prominent role in brain function and disease, we hypothesized that microRNAs (miRNAs) might be of importance for BD. Here we show that levels of miR-34a, which is predicted to target multiple genes implicated as genetic risk factors for BD, are increased in postmortem cerebellar tissue from BD patients, as well as in BD patient-derived neuronal cultures generated by reprogramming of human fibroblasts into induced neurons or into induced pluripotent stem cells (iPSCs) subsequently differentiated into neurons. Of the predicted miR-34a targets, we validated the BD risk genes ankyrin-3 (ANK3) and voltage-dependent L-type calcium channel subunit beta-3 (CACNB3) as direct miR-34a targets. Using human iPSC-derived neuronal progenitor cells, we further show that enhancement of miR-34a expression impairs neuronal differentiation, expression of synaptic proteins and neuronal morphology, whereas reducing endogenous miR-34a expression enhances dendritic elaboration. Taken together, we propose that miR-34a serves as a critical link between multiple etiological factors for BD and its pathogenesis through the regulation of a molecular network essential for neuronal development and synaptogenesis.

Cis-acting regulation of brain-specific ANK3 gene expression by a genetic variant associated with bipolar disorder.

Several genome-wide association studies for bipolar disorder (BD) have found a strong association of the Ankyrin 3 (ANK3) gene. This association spans numerous linked single-nucleotide polymorphisms (SNPs) in an ~250-kb genomic region overlapping ANK3. The associated region encompasses predicted regulatory elements as well as two of the six validated alternative first exons, which encode distinct protein domains at the N-terminus of the protein also known as Ankyrin-G. Using RNA ligase-mediated rapid amplification of cDNA ends to identify novel transcripts in conjunction with a highly sensitive, exon-specific multiplexed mRNA expression assay, we detected differential regulation of distinct ANK3 transcription start sites and coupling of specific 5' ends with 3' mRNA splicing events in postmortem human brain and human stem cell-derived neural progenitors and neurons. Furthermore, allelic variation at the BD-associated SNP rs1938526 correlated with a significant difference in cerebellar expression of a brain-specific ANK3 transcript. These findings suggest a brain-specific cis-regulatory transcriptional effect of ANK3 that may be relevant to BD pathophysiology.

Activation and repression of transcription initiation by a distant DNA structural transition.

Negative superhelical tension can drive local transitions to alternative DNA structures. Long regions of DNA may contain several sites that are susceptible to forming alternative structures. Their relative propensities to undergo transition are ordered according to the energies required for their formation. These energies have two components - the energy needed to drive the transition and the energy relieved by the partial relaxation of superhelicity that the transition provides. This coupling can cause a complex competition among the possible transitions, in which the formation of one energetically favourable alternative structure may inhibit the formation of another within the same domain. In principle, DNA structural competitions can affect the structural and energetic requirements for the initiation of transcription at distant promoter sites. We have tested this possibility by examining the effects of structural transitions on transcription initiation from promoter sites in the same superhelical domain. Specifically, we describe the effects of the presence of a Z-DNA-forming DNA sequence on the basal levels of expression of two supercoiling-sensitive promoters of Escherichia coli, ilvPG and gyrA. We demonstrate transcriptional repression of the ilvPG promoter and activation of the gyrA promoter. We present evidence that this regulation is effected by the superhelically induced B- to Z-DNA transition in a manner that is both orientation and distance independent. We discuss the mechanism of topological coupling between left-handed Z-DNA and the regulation of promoter activity. We also discuss the possibility that the coupling of DNA structural transitions and transcriptional activity might be used as a general regulatory mechanism for gene expression.

Inhibition of DNA supercoiling-dependent transcriptional activation by a distant B-DNA to Z-DNA transition.

Negative DNA superhelicity can destabilize the local B-form DNA structure and can drive transitions to other conformations at susceptible sites. In a molecule containing multiple susceptible sites, superhelicity can couple these alternatives together, causing them to compete. In principle, these superhelically driven local structural transitions can be either facilitated or inhibited by proteins that bind at or near potential transition sites. If a DNA region that is susceptible to forming a superhelically induced alternate structure is stabilized in the B-form by a DNA-binding protein, its propensity for transition will be transferred to other sites within the same domain. If one of these secondary sites is in a promoter region, this transfer could facilitate open complex formation and thereby activate gene expression. We previously proposed that a supercoiling-dependent, DNA structural transmission mechanism of this type is responsible for the integration host factor-mediated activation of transcription from the ilvPG promoter of Escherichia coli (Sheridan, S. D., Benham, C. J. & Hatfield, G. W. (1998) J. Biol. Chem. 273, 21298-21308). In this report we confirm the validity of this mechanism by demonstrating the ability of a distant Z-DNA-forming site to compete with the superhelical destabilization that is required for integration host factor-mediated transcriptional activation, and thereby delay its occurrence.

Activation of gene expression by a novel DNA structural transmission mechanism that requires supercoiling-induced DNA duplex destabilization in an upstream activating sequence.

We have previously demonstrated that integration host factor (IHF)-mediated activation of transcription from the ilvPG promoter of Escherichia coli requires a supercoiled DNA template and occurs in the absence of specific interactions between IHF and RNA polymerase. In this report, we describe a novel, supercoiling-dependent, DNA structural transmission mechanism for this activation. We provide theoretical evidence for a supercoiling-induced DNA duplex destabilized (SIDD) structure in the A + T-rich, ilvPG regulatory region between base pair positions +1 and -160. We show that the region of this SIDD sequence immediately upstream of an IHF binding site centered at base pair position -92 is, in fact, destabilized by superhelical stress and that this duplex destabilization is inhibited by IHF binding. Thus, in the presence of IHF, the negative superhelical twist normally absorbed by this DNA structure in the promoter distal half of the SIDD sequence is transferred to the downstream portion of the SIDD sequence containing the ilvPG promoter site. This IHF-mediated translocation of superhelical energy facilitates duplex destabilization in the -10 region of the downstream ilvPG promoter and activates transcription by increasing the rate of open complex formation.

Effects of integration host factor and DNA supercoiling on transcription from the ilvPG promoter of Escherichia coli.

Integration host factor (IHF) activates transcription from the ilvPG promoter by severely distorting the DNA helix in an upstream region of a supercoiled DNA template in a way that alters the structure of the DNA in the downstream promoter region and facilitates open complex formation. In this report, the in vivo and in vitro influence of DNA supercoiling on transcription from this promoter is examined. In the absence of IHF, promoter activity increases with increased DNA supercoiling. In the presence of IHF, the same increases in superhelical DNA densities result in larger increases in promoter activity until a maximal activation of 5-fold is obtained. However, the relative transcriptional activities of the promoter in the presence and absence of IHF at any given DNA superhelical density remains the same. Thus, IHF and increased DNA supercoiling activate transcription by different mechanisms. Also, IHF binds with equal affinities to its target site on linear and supercoiled DNA templates. Therefore, IHF binding does not activate transcription simply by increasing the local negative supercoiling of the DNA helix in the downstream promoter region or by differential binding to relaxed and supercoiled DNA templates.