Introduction
============

Amyotrophic lateral sclerosis (ALS) is an intractable neurodegenerative disease, characterized by rapid and progressive degeneration of motor neurons in the cerebral cortex and the spinal cord. Approximately 10% of ALS is of familial origin caused by genetic mutations of superoxide dismutase 1 (SOD1), TAR DNA binding protein (TARDBP, TDP-43), fused in sarcoma (FUS), optineurin (OPTN), ubiquilin 2 (UBQLN2), and chromosome 9 open reading frame 72 (C9orf72).[@b1-jcnsd-7-2015-001] At present, the precise molecular mechanism underlying motor neuron degeneration in sporadic ALS remains unknown. Increasing evidence indicates that dysfunction of oligodendrocytes plays a key role in pathological processes of ALS. The levels of expression of oligodendrocyte-specific monocarboxylate transporter 1, which transports lactate, an essential energy metabolite to motor neurons, are greatly reduced in the motor cortex of ALS patients and the spinal cord of SOD1 mutant mice.[@b2-jcnsd-7-2015-001] In the spinal cord of ALS patients, TDP-43-positive inclusions accumulate not only in motor neurons but also in oligodendrocytes.[@b3-jcnsd-7-2015-001] Extensive degeneration of gray matter oligodendrocytes is found in the motor cortex and the spinal cord of ALS patients.[@b4-jcnsd-7-2015-001] Furthermore, genetic deletion of mutant SOD1 selectively from oligodendrocytes markedly ameliorates the disease course in SOD1 mutant mice.[@b4-jcnsd-7-2015-001]

The basic helix--loop--helix (bHLH) transcription factor Olig2, a downstream target of a ventralizing factor Sonic hedgehog (SHH) that is secreted from the notochord and floor plate, plays a pivotal role in the development and differentiation of both motor neurons and oligodendrocytes in the spinal cord.[@b5-jcnsd-7-2015-001] Olig2 is expressed in a restricted domain of the spinal cord ventricular zone named progenitor of motor neuron (pMN), which is composed of a pool of progenitor cells that sequentially generate motor neurons and oligodendrocytes, supporting evidence for the shared motor neuron/oligodendrocyte lineage.[@b6-jcnsd-7-2015-001],[@b7-jcnsd-7-2015-001] In contrast, V0, V1, V2, and V3 interneurons are separately generated from the p0, p1, p2, and p3 domains, respectively.[@b8-jcnsd-7-2015-001] The pMN domain is bounded ventrally by the p3 domain defined by the expression of NK2 homeobox 2 (Nkx2.2, Nkx2--2), a homeodomain transcription factor, and dorsally by the p2 domain, whose ventral boundary is defined by the expression of Iroquois related homeobox 3 (Irx3), another homeodomain protein.[@b6-jcnsd-7-2015-001] Both motor neurons and oligodendrocytes are almost completely lost in mice with a homozygous inactivation of Olig2.[@b9-jcnsd-7-2015-001] Olig2 specifies the pMN identity by direct repression of interneuron transcription programs.[@b10-jcnsd-7-2015-001] Notably, in Oligo1/2 double-mutant mice, pMN progenitors generate V2 interneurons and astrocytes.[@b11-jcnsd-7-2015-001] A microRNA mir-17--3p silences the expression of Olig2 in the p2 domain to generate V2 interneurons.[@b12-jcnsd-7-2015-001] Olig2 plays a decisive role in the specification of the pMN domain and initiates motor neuron differentiation by inducing expression of a proneural bHLH factor neurogenin 2 (Neurog2, Ngn2), which drives pMN progenitors to leave the cell cycle and express differentiation markers characteristic of post-mitotic neurons.[@b13-jcnsd-7-2015-001] Ngn2 expression is extinguished from the progeny destined to become oligodendrocytes, whereas the expression of Olig2 is downregulated in the progeny destined to become motor neurons.[@b14-jcnsd-7-2015-001] In contrast, the expression of Nkx2.2 is upregulated in pMN progenitors as they switch from producing motor neurons to generating oligodendrocytes.[@b15-jcnsd-7-2015-001]

Structurally, Olig2 is characterized by the presence of the bHLH domain that mediates dimerization and binding to target DNA sequences, often containing the core hexanucleotide motif composed of CANNTG named the E-box.[@b5-jcnsd-7-2015-001] The bHLH domains of Olig1 and Olig2 exhibit more than 80% amino acid identities. However, Olig1 and Olig2 exert non-overlapping biological functions attributable to differential interactions with dimeric partners and co-regulator proteins.[@b5-jcnsd-7-2015-001] Olig2 favors forming a homodimer, while Ser147 phosphorylation of Olig2 by protein kinase A induces motor neuron differentiation by switching of dimerization partner from Olig2 to Ngn2.[@b16-jcnsd-7-2015-001] The levels of Olig2 expression in the developing spinal cord are much higher than those of Olig1, and Olig2 has a broader expression pattern in the embryonic forebrain.[@b9-jcnsd-7-2015-001] Furthermore, deletion of Olig1 has no visible impact on the generation of motor neurons and early oligodendrocyte progenitors.[@b5-jcnsd-7-2015-001],[@b17-jcnsd-7-2015-001] Olig2 generally acts as a transcriptional repressor of direct target genes.[@b5-jcnsd-7-2015-001],[@b13-jcnsd-7-2015-001] However, at present, the complete profile of Olig2 target genes in both pMNs and oligodendrocyte progenitor cells (OPCs) remains to be intensively characterized, particularly with relevance to the pathogenesis of ALS.

Recently, an advanced next-generation sequencing (NGS) technology termed chromatin immunoprecipitation, followed by deep sequencing (ChIP-Seq), encouraged us to characterize genome-wide profiles of DNA-binding proteins and histone modifications.[@b18-jcnsd-7-2015-001] ChIP-Seq, with the advantages of higher resolution, less noise, and greater coverage of the genome compared with microarray-based ChIP-Chip, serves as an innovative tool for studying gene regulatory networks on the whole-genome scale. To investigate a role of Olig2 in the pathogenesis of ALS, we attempted to characterize the comprehensive set of ChIP-Seq-based Olig2 direct target genes in pMNs and OPCs and their molecular networks by analyzing datasets retrieved from the public database.

Methods
=======

ChIP-Seq dataset
----------------

We retrieved an Olig2 ChIP-Seq dataset of embryonic stem cells (ESCs) during motor neuron differentiation from the DDBJ Sequence Read Archive (DRA) (trace.ddbj.nig.ac.jp/DRASearch) under the accession number of SRP007566. The original experiment was carried out by the researchers in Dr. Hynek Wichterle's Laboratory, Columbia University Medical Center.[@b10-jcnsd-7-2015-001] The relevant dataset numbered GSE30882 is open to public since November 12, 2011. They generated a mouse ESC line incorporated with a transgene composed of doxycycline (DOX)-inducible Olig2 tagged with the V5 epitope in the carboxyl terminal, named iOlig2-V5. Then, motor neuron differentiation was induced by exposure of embryoid bodies derived from ESCs to 1 μM all-trans retinoic acid (RA, Sigma) and 0.5 μM Smoothened Agonist (SAG, Merck/Calbiochem), an agonist of hedgehog signaling. This was followed by a 24-hour treatment with 1 μg/mL DOX starting on day 3, resulting in differentiation of motor neurons progenitors (pMNs) expressing an Olig2-V5 fusion protein, along with endogenous Olig2 (Olig2-V5^+^pMNs). In some experiments, pMN cells untreated with DOX (Olig2-V5^−^ pMNs) were utilized to detect binding sites for an endogenous Olig2 protein. The cells were fixed with formaldehyde for ChIP. Sonicated nuclear extracts were immunoprecipitated with a rabbit polyclonal anti-Olig2 antibody (AB15328, Mil-lipore) for ChIP of Olig2-V5^−^ pMNs (SRR315585), a rabbit polyclonal anti-V5 antibody (ab15828, Abcam) for ChIP of Olig2-V5^+^ pMNs (SRR315586), or with ab15828 for ChIP of Olig2-V5^−^ pMNs serving as a negative control (SRR315590). NGS libraries constructed from adapter-ligated ChIP DNA fragments were processed for deep sequencing on Genome Analyzer IIx (Illumina).

To compare Olig2 target genes in pMNs with those in OPCs, we retrieved another Olig2 ChIP-Seq dataset from DRA under the accession number of SRP015333. The original experiment was carried out by the researchers in Dr. Qing Richard Lu's Laboratory, University of Texas Southwestern Medical Center.[@b19-jcnsd-7-2015-001] The relevant dataset numbered GSE40506 is open to public since August 31, 2012. In their experiment, purified OPCs isolated from newborn rat cerebral cortices at P2 were grown in the OPC growth medium containing 10 ng/mL platelet-derived growth factor (PDGF)-AA and 20 ng/mL basic fibroblast growth factor (bFGF). Then, they were incubated for 3 days in the oligodendrocyte differentiation medium containing 15 nM triiodothyronine (T3) and 10 ng/mL ciliary neurotrophic factor (CNTF). The cells were fixed with formaldehyde for ChIP. Sonicated nuclear extracts were immunoprecipitated with a rabbit polyclonal anti-Olig2 antibody (ab136253, Abcam) for ChIP of the endogenous Olig2 protein expressed in differentiating oligodendrocytes (SRR548313), or with rabbit anti-human IgG (ab2410, Abcam) serving as a negative control (SRR548314). NGS libraries constructed from adapter-ligated ChIP DNA fragments were processed for deep sequencing on Genome Analyzer IIx.

RNA-Seq datasets
----------------

To study the involvement of Olig2 target genes in motor neuron degeneration *in vivo* in an animal model of ALS, we first retrieved a RNA-Seq dataset from DRA under the accession number of SRP013849. The original experiment was carried out by the researchers in Dr. Arthur L. Horwich's Laboratory, Yale University School of Medicine.[@b20-jcnsd-7-2015-001] The relevant dataset numbered GSE38820 is open to public since December 4, 2012. In their experiment, motor neuron cell bodies were isolated by laser-captured microdissection (LCM) from spinal cords of transgenic mouse strains at 3 months of age in the presymptomatic stage, including wild-type SOD1-YFP (strain 592; SRR515121 and SRR515122) and mutant G85R SOD1-YFP (strain 737; SRR515123 and SRR515124). Total RNA extracted from pooled motor neuron cell bodies was subjected to polyA selection, fragmentation, cDNA synthesis, adaptor ligation, and library amplification. NGS libraries were processed for single-end sequencing on Genome Analyzer IIx.

To verify a pivotal role of Olig2 target genes *in vivo* in ALS patients, next we retrieved another RNA-Seq dataset numbered SRP033464 from DRA. The original experiment was carried out by the researchers in Dr. Oleg Butovsky's Laboratory, Brigham and Women's Hospital, Harvard Medical School.[@b21-jcnsd-7-2015-001] The relevant dataset numbered GSE52946 is open to public since November 24, 2014. In their experiment, whole lumbar spinal cord tissues were isolated from eight sporadic and two familial ALS (SOD1 A4V) patients and 10 healthy control subjects according to the guideline approved by the Institutional Review Boards at Massachusetts General Hospital and Brigham and Women's Hospital. Total RNA was extracted by using mirVana^™^ miRNA isolation kit (Ambion) according to the manufacturer's protocol. Paired-end RNA-Seq analysis was performed on HiSeq 2000 (Illumina) in the Harvard genetic facility.

NGS data analysis
-----------------

First, we evaluated the quality of NGS short reads by searching on the FastQC program ([www.bioinformatics.babraham.ac.uk/projects/fastqc](http://www.bioinformatics.babraham.ac.uk/projects/fastqc)). Then, we removed the reads of insufficient quality by filtering them out by the FASTX toolkit ([hannonlab.cshl.edu/fastx_toolkit](http://hannonlab.cshl.edu/fastx_toolkit)). For ChIP-Seq analysis, we mapped the cleaned data on the mouse genome reference sequence version mm9 or the rat genome reference sequence version rn4 by a mapping tool named COBWeb of the Strand NGS2.0 program, formerly named Avadis NGS (Strand Genomics) or by Bowtie version 1.0.0 or 2.1.0 ([bowtie-bio.sourceforge.net](http://bowtie-bio.sourceforge.net)). Then, we identified the peaks of the binding sites by using the Model-based Analysis of ChIP-Seq (MACS) program, as described previously.[@b22-jcnsd-7-2015-001] We determined the genes corresponding to the peaks by a neighboring gene analysis tool of Strand NGS in the setting within a distance of 5000 bp from peaks to genes. We identified the consensus motif sequences surrounding the peaks by using the GADEM program[@b23-jcnsd-7-2015-001] and the genomic location of binding peaks by a peak-finding tool of Strand NGS. We imported the processed data into a genome viewer named GenomeJack v1.4 (Mitsubishi Space Software).[@b22-jcnsd-7-2015-001] For RNA-Seq analysis, after removing poly-A tails and low-quality reads from NGS short-read data, we mapped them on mm9 or hg19 by TopHat version 2.0.9 ([ccb.jhu.edu/software/tophat/index.shtml](http://ccb.jhu.edu/software/tophat/index.shtml)), and identified differentially expressed genes that satisfied the significance expressed as *Q*-value (FDR-adjusted *P*-value) ≤0.01 by Cufflinks version 2.1.1 ([cufflinks.cbcb.umd.edu](http://cufflinks.cbcb.umd.edu)).

Molecular network analysis
--------------------------

To identify molecular networks biologically relevant to ChIP-Seq-based Olig2 target genes, we imported the corresponding Entrez Gene IDs into the Functional Annotation tool of Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.7 ([david.abcc.ncifcrf.gov](http://david.abcc.ncifcrf.gov)).[@b24-jcnsd-7-2015-001] DAVID identifies relevant pathways constructed by the Kyoto Encyclopedia of Genes and Genomes (KEGG), composed of the genes enriched in the given set, followed by statistical evaluation by a modified Fisher's exact test after correction by Bonferroni multiple comparison test. KEGG ([www.kegg.jp](http://www.kegg.jp)) is a publicly accessible knowledge base that covers a wide range of pathway maps on metabolic, genetic, environmental, and cellular processes, as well as human diseases, and is currently composed of 381,274 pathways generated from 473 reference pathways.[@b25-jcnsd-7-2015-001]

We also imported Entrez Gene IDs into Ingenuity Pathways Analysis (IPA) (Ingenuity Systems; [www.ingenuity.com](http://www.ingenuity.com)). IPA is a commercial knowledge base that contains approximately 3,000,000 biological and chemical interactions and functional annotations with definite scientific evidence. By uploading the list of Gene IDs, the network-generation algorithm identifies focused genes integrated in global molecular pathways and networks in the setting of 70 molecules per network. IPA calculates the score *P*-value that reflects the statistical significance of association between the genes and the pathways or networks by the Fisher's exact test.

KeyMolnet (KM Data; [www.km-data.jp](http://www.km-data.jp)), another commercial knowledge base, contains manually curated contents on 164,000 relationships among human genes and proteins, small molecules, diseases, pathways, and drugs.[@b26-jcnsd-7-2015-001] They include the core contents collected from selected review articles with the highest reliability. By importing the list of Gene ID, KeyMolnet automatically provides the corresponding molecules as nodes on the network. The neighboring network-search algorithm selected one or more molecules as starting points to generate the network of all kinds of molecular interactions around starting molecules, including direct activation/inactivation, transcriptional activation/repression, and the complex formation within one path from the starting points. The generated network was compared side by side with 501 human canonical pathways of the KeyMolnet library. The algorithm, which counts the number of overlapping molecular relations between the extracted network and the canonical pathway, makes it possible to identify the canonical pathway showing the most significant contribution to the extracted network.

Results
=======

Identification of 5966 ChIP-Seq-based Olig2 target genes in motor neuron progenitor cells
-----------------------------------------------------------------------------------------

First, we evaluated the quality of ChIP-Seq data reanalyzed in the present study. After cleaning short-read data, the quality scores exceeded 20 across the bases on FastQC, indicating an acceptable quality of the data for downstream analysis ([Supplementary Fig. 1](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). After mapping cleaned data on mm9 by COBWeb, we identified 43,419 ChIP-Seq peaks for the binding sites of the endogenous Olig2 protein and 24,112 ChIP-Seq peaks for binding sites of the Olig2-V5 fusion protein in mouse ESC-derived pMNs progenitors. We extracted the set of 20,043 peaks shared between both as the most reliable set of Olig2 ChIP-Seq peaks. From these, we collected the peaks located within a distance of 5000 bp from protein coding genes. Finally, we identified a set of 5966 Olig2 target genes that satisfied fold enrichment (FE) ≥10 and false discovery rate (FDR) ≤0.1 ([Supplementary Table 1](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Importantly, the list contained previously reported Olig2 direct targets,[@b10-jcnsd-7-2015-001],[@b19-jcnsd-7-2015-001] such as Nkx2.2 (FE = 353.7) ([Fig. 1](#f1-jcnsd-7-2015-001){ref-type="fig"}), Zeb2 (Sip1, FE = 88.5), Pax6 (FE = 87.4), and Irx3 (FE = 24.8). Furthermore, we identified Trp53 (FE = 108.4) and Ngn2 (EF = 28.6) as a category of unreported Olig2 targets ([Supplementary Table 1](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). The peaks were distributed in the upstream (12.9%), 5′UTR (11.8%), 3′UTR (2.0%), coding exonic (13.1%), and intronic (54.3%) regions. The motif analysis by GADEM revealed a relative enrichment of the E-box consensus sequence defined as 5′-CAGCTG-3′ located within genomic regions surrounding ChIP-Seq peaks ([Fig. 1](#f1-jcnsd-7-2015-001){ref-type="fig"} and [Supplementary Fig. 2](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}).

Identification of 1553 ChIP-Seq-based Olig2 target genes in OPCs
----------------------------------------------------------------

Next, we studied Olig2 target genes in rat OPCs by mapping ChIP-Seq data on rn4 by COBWeb. We identified 10,756 ChIP-Seq peaks in OPCs upon differentiation into oligodendrocytes. From these, we extracted the peaks located within a distance of 5000 bp from protein coding genes. Finally, we identified a set of 1553 Olig2 target genes that satisfied FE ≥10 and FDR ≤0.1 ([Supplementary Table 2](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). The peaks were distributed in the upstream (9.2%), 5′UTR (11.8%), 3′UTR (1.3%), coding exonic (10.4%), and intronic (59.5%) regions. Importantly, the set of 740 genes among them were overlapped between pMNs and OPCs ([Supplementary Table 3](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}, underlined in [Supplementary Tables 1](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"} and [2](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Importantly, functional annotation analysis by DAVID showed that 320 genes (43.2%) out of the set of 740 Olig2 target genes overlapping between pMNs and OPCs were significantly related to "alternative splicing" in the Swiss-Prot (SP)/Protein Information Resource (PIR) keyword (*P* = 3.73E--31 corrected by Bonferonni; underlined in [Supplementary Table 3](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}).

Molecular networks of ChIP-Seq-based Olig2 target genes in motor neuron progenitor cells and OPCs
-------------------------------------------------------------------------------------------------

Next, we studied molecular networks of Olig2 target genes by using three distinct pathway analysis tools of bioinformatics. By using DAVID, we identified functionally associated gene ontology (GO) terms. For 5966 Olig2 targets in pMNs, the most significant GO terms included "cell morphogenesis" (GO:0000902; *P* = 3.17E--16 corrected by Bonferroni) for biological process, "plasma membrane" (GO:0005886; *P* = 5.54E--18) for cellular component, and "metal ion binding" (GO:0046872; *P* = 6.46E--20) for molecular function. For 1533 Olig2 targets in OPCs, the most significant GO terms included "intracellular signaling cascade" (GO:0007242; *P* = 2.53E--05) for biological process, "plasma membrane" (GO:0005886; *P* = 1.13E--05) for cellular component, and "ion binding" (GO:0043167; *P* = 3.07E--04) for molecular function.

By KEGG pathway analysis, the set of 5966 Olig2 targets in pMNs showed a significant relationship with top three pathways defined as "Pathways in cancer" (mmu05200; *P* = 5.08E--08 corrected by Bonferonni), "Axon guidance" (mmu04360; *P* = 1.76E--09) ([Fig. 2](#f2-jcnsd-7-2015-001){ref-type="fig"}), and "Focal adhesion" (mmu04510; *P* = 2.90E--07) ([Table 1](#t1-jcnsd-7-2015-001){ref-type="table"}). Importantly, they also exhibited significant association with the pathways defined as "Glioma" (mmu05214; rank 17, *P* = 0.024) ([Supplementary Fig. 3](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}) and "Hedgehog signaling pathway" (mmu04340; rank 18, *P* = 0.033), where patched homolog 1 (Ptch1), the receptor for SHH, corresponded to one of Olig2 targets ([Table 1](#t1-jcnsd-7-2015-001){ref-type="table"}). From the 5966 genes described above, we extracted the set of 4717 Olig2 target genes that satisfied 10 times more stringent FDR (FDR ≤0.01) ([Supplementary Table 4](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). We imported them into the Functional Annotation tool of DAVID to identify relevant KEGG pathways. Again, we found that the set of 4717 Olig2 target genes in pMN showed the most significant relationship with "Axon guidance" (mmu04360; *P* = 1.76E--09 corrected by Bonferroni), "Pathways in cancer" (mmu05200; *P* = 4.82E--09), "MAPK signaling pathway" (mmu04010; *P* = 7.11E--06), and "Focal adhesion" (mmu04510; *P* = 2.73--04), validating the reliability of initial results. For the 1533 Olig2 targets in OPCs, the top three KEGG pathways included "MAPK signaling pathway" (rno04010; *P* = 1.49E--05 corrected by Bonferroni), "Long-term depression" (rno04730; *P* = 3.34E--05), and "Vascular smooth muscle contraction" (rno04270; *P* = 3.57E--03).

Next, we studied molecular networks of 5966 Olig2 targets in pMNs by using the Core Analysis tool of IPA. They showed a significant relationship with canonical pathways defined as "Axonal guidance signaling" (*P* = 3.70E--17) and "Molecular mechanisms of cancer" (*P* = 3.64E--15), well consistent with the results of KEGG. For the 1533 Olig2 targets in OPCs, they exhibited a significant relationship with canonical pathways defined as "Synaptic long term depression" (*P* = 2.98E--07) and "Wnt/β-catenin signaling" (*P* = 3.65E--06).

IPA also identified functional networks highly relevant to the 5966 Olig2 targets in pMNs, defined as "Cancer, cell-to-cell signaling and interaction, cellular assembly and organization" (*P* = 1.00E--54), "Auditory disease, connective tissue disorders, dermatological diseases and conditions" (*P* = 1.00E--54), "Cancer, gastrointestinal disease, organismal injury and abnormalities" (*P* = 1.00E--52), and "RNA post-transcriptional modification, embryonic development, hair and skin development and function" (*P* = 1.00E--52), where both Cul1 (cullin 1) and Cul3 (cullin 3), which constitute the CULLIN-RING ubiquitin ligase (CRL) complex, play a central role in the Olig2 target gene network ([Fig. 3](#f3-jcnsd-7-2015-001){ref-type="fig"}). For the 1533 Olig2 targets in OPCs, IPA identified a functional network defined as "Cell death and survival, cellular compromise, neurological disease" (*P* = 1.00E--83) as the top rank network.

Next, we studied molecular networks of the 5966 Olig2 targets in pMNs by using KeyMolnet. The neighboring network-search algorithm extracted the extremely complex network composed of 5839 molecules and 13,818 molecular relations ([Supplementary Fig. 4](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). The network showed the most significant relationship with "Transcriptional regulation by p53" (*P* = 1.68E--237). For the 1533 Olig2 targets in OPCs, KeyMolnet identified the highly complex network showing the most significant relationship with "Transcriptional regulation by NF-κB" (*P* = 1.28E--181) (data not shown).

Downregulation of numerous Olig2 target genes in degenerating motor neurons of mutant SOD1 transgenic mice and spinal cord tissues of ALS patients
--------------------------------------------------------------------------------------------------------------------------------------------------

Finally, we studied a possible role of Olig2 target genes in the process of motor neuron degeneration in a mouse model of ALS and lumbar spinal cord tissues of ALS patients. We identified the set of 277 genes downregulated in LCM-purified spinal cord motor neurons derived from presymptomatic SOD1 G85R transgenic mice, which satisfied a *Q*-value ≤0.01 and log2 fold change ≤--1.0 ([Supplementary Table 5](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Among them, the set of 77 genes (27.8%) corresponded to Olig2 target genes in pMNs ([Table 2](#t2-jcnsd-7-2015-001){ref-type="table"}, underlined in [Supplementary Table 5](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). We also identified the set of 1583 genes downregulated in lumbar spinal cord tissues of ALS patients, which satisfied a *Q*-value ≤0.01 and log2 fold change ≤--1.0 ([Supplementary Table 6](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Among them, the set of 473 genes (29.9%) corresponded to Olig2 target genes in pMNs (underlined in [Supplementary Table 6](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Since Olig2 generally acts as a transcriptional repressor of direct target genes,[@b5-jcnsd-7-2015-001],[@b13-jcnsd-7-2015-001] these results suggest that downregulation of Olig2 target genes possibly due to overactivation of Olig2 plays a key role in progression of motor neuron degeneration both in mutant SOD mice and in ALS patients.

Discussion
==========

ALS is a fatal neurodegenerative disease that chiefly affects motor neurons in the brain and spinal cord. All motor neurons in the spinal cord arise from a common set of progenitor cells located within the pMN domain.[@b6-jcnsd-7-2015-001],[@b7-jcnsd-7-2015-001] Recent studies have convincingly indicated an active involvement of oligodendrocyte dysfunction in the pathogenesis of ALS.[@b2-jcnsd-7-2015-001]--[@b4-jcnsd-7-2015-001] Olig2, expressed selectively by pMN progenitor cells, plays an indispensable role in the development and differentiation of both motor neurons and oligodendrocytes in the spinal cord.[@b5-jcnsd-7-2015-001]--[@b9-jcnsd-7-2015-001] Here, we characterized the comprehensive set of ChIP-Seq-based Olig2 direct target genes and their molecular networks in pMNs and OPCs with relevance to the pathogenesis of ALS.

First, we identified a reproducible set of 5966 Olig2 target genes in pMNs from the ChIP-Seq dataset numbered SRP007566. They included previously reported Olig2 direct targets in pMNs, such as Nkx2.2, Pax6, and Irx3,[@b10-jcnsd-7-2015-001] supporting the validity of our results. Next, we identified the cardinal set of 1553 Olig2 target genes in OPCs from the ChIP-Seq dataset numbered SRP015333. Then, we extracted the core set of 740 Olig2 target genes overlapping between pMNs and OPCs. Importantly, the genes closely related to "alternative splicing" in the category of SP/PIR keywords are highly enriched in Olig2 targets shared between pMNs and OPCs. Increasing evidence indicates that RNA metabolism, including regulation of transcription and alternative splicing, is profoundly disturbed in ALS.[@b27-jcnsd-7-2015-001],[@b28-jcnsd-7-2015-001] Notably, TDP-43 (TARDBP) and FUS, both of which are causative genes of familial ALS and play a key role in RNA processing and regulation of exon splicing, are mislocated from the nucleus to the cytoplasm in degenerating motor neurons of ALS.[@b29-jcnsd-7-2015-001] Most importantly, we found that approximately one-third of the downregulated genes in LCM-purified spinal cord motor neurons of presymptomatic SOD1 G85R transgenic mice, as well as in lumbar spinal cord tissues of ALS patients, correspond to Olig2 targets in pMNs, suggesting an involvement of overactivation of Olig2 in the pathogenesis of ALS, beginning from the very early stage of the disease.

We intensively studied molecular networks of Olig2 target genes in pMNs and OPCs. By analyzing with KEGG and IPA, the set of 5966 Olig2 targets in pMNs showed a significant relationship with "Molecular mechanisms and pathways in cancer" and "Axon guidance signaling", while 1533 Olig2 target genes in OPCs exhibited relevance to "Synaptic long term depression", "MAPK signaling pathway", and "Wnt/β-catenin signaling". Interestingly, synaptic plasticity regulated by a balance between long-term depression and potentiation is altered in the brain of SOD G93A transgenic mice.[@b30-jcnsd-7-2015-001] Conditional ablation of Erk1/2 in oligodendrocytes in the adult CNS reduces the expression of a key transcription factor named the myelin gene regulatory factor (Myrf), which is indispensable for myelin gene expression.[@b31-jcnsd-7-2015-001] Protein levels of Wnt4 and Wnt inhibitory factor-1 (Wif1) are elevated in the spinal cord of SOD1 G93A mice.[@b32-jcnsd-7-2015-001] Smad-interacting protein-1 (Sip1; Zeb2) activates Smad7 that antagonizes the bone morphogenetic protein (BMP) and Wnt/β-catenin signaling pathways pivotal for the development of astrocytes.[@b19-jcnsd-7-2015-001] Olig2 binds to the motor neuron and pancreas homeobox 1 (Mnx1, Hb9) promoter and represses its transcription to keep the replication-competent state of pMNs during neural tube development.[@b14-jcnsd-7-2015-001] Olig2 binds to the promoter of cyclin-dependent kinase inhibitor 1A (Cdkn1a, p21) and directly suppresses its expression.[@b33-jcnsd-7-2015-001] We identified Zeb2 (Sip1), Mnx1, and Cdkn1a as Olig2 targets in pMNs. These results suggest that Olig2 downregulates a wide range of target genes involved in diverse neuronal and glial functions under physiological conditions during development to avoid premature differentiation of Olig2-expressing cells and under pathological conditions to suppress oncogenesis of these cells. However, Olig2 is expressed in not only normal mature oligodendrocytes but also oligodendroglioma and diffuse glioma irrespective of their grades.[@b34-jcnsd-7-2015-001] Furthermore, we found a close relationship between Olig2 targets in pMNs and KEGG "Glioma" pathway. The apparent discrepancies might be derived from the proliferative functions of Olig2 affected by post-transcriptional modifications, which are largely controlled in neural progenitor cells by developmentally regulated phosphorylation of a triplet serine motif of Ser10, Ser13, and Ser14 located on the N-terminus of Olig2.[@b35-jcnsd-7-2015-001] Olig2, phosphorylated at the triplet serine motif and expressed in p53-positive human gliomas, inhibits biological responses to p53.[@b36-jcnsd-7-2015-001] Interestingly, KeyMolnet indicated that the molecular network of Olig2 targets in pMNs is the most relevant to the network of "Transcriptional regulation by p53".

The ubiquitin-proteasome system, pivotal for protein turnover to prevent accumulation of abnormal proteins in the cell, is severely compromised in ALS.[@b37-jcnsd-7-2015-001] IPA showed that Olig2 target genes in pMNs have relevance to the functional network defined as "RNA post-transcriptional modification, embryonic development, hair and skin development and function", where Cul1 and Cul3, both of which are central components of the CRL complex involved in regulation of protein quality control, serve as a hub in the Olig2 target gene network. These observations suggest the possibility that even subtle overactivation of Olig2 could induce a serious defect in physiological function of the CRL complex essential for cellular protein homeostasis, leading to neurodegeneration due to accumulation of disease-associated proteins.[@b38-jcnsd-7-2015-001]

Conclusions
===========

By reanalyzing ChIP-Seq datasets, we identified the set of 5966 Olig2 target genes in pMNs and the set of 1553 Olig2 target genes in OPCs. The genes related to the SP/PIR keyword "alternative splicing" are clustered in the core set of 740 targets overlapping between pMNs and OPCs. Approximately one-third of downregulated genes in purified motor neurons of presymptomatic mutant SOD1 transgenic mice and in lumbar spinal cord tissues of ALS patients correspond to Olig2 target genes in pMNs. Molecular network analysis suggests that Olig2 downregulates a wide range of target genes involved in diverse neuronal and glial functions. We identified a novel Olig2 target gene network composed of the CRL complex, which is possibly involved in clearance of disease-associated proteins. These observations lead to a novel hypothesis that aberrant regulation of Olig2 function, by affecting biology of both motor neurons and oligodendrocytes, might be involved in the pathogenesis of ALS.

Supplementary Data
==================

######

**Supplementary Figure 1.** FastQC profile of five ChIP-Seq data analyzed in the present study. FASTQ format files of cleaned short read NGS data derived from SRR315585 (panel a), SRR315586 (panel b), SRR315590 (panel c), SRR548313 (panel d), and SRR548314 (panel e) were imported into the FastQC program. The per-base sequence quality score is shown with the median (red line), the mean (blue line), and the interquartile range (yellow box).

**Supplementary Figure 2.** Olig2-binding consensus sequence motif. The consensus motif sequence surrounding Olig2 ChIP-Seq peaks was identified by GADEM. The canonical E-box consensus sequence defined as 5′-CANNTG-3′ were found on 2376 peaks among a total of 20,043 peaks in pMNs detected by MACS.

**Supplementary Figure 3.** KEGG "Glioma" pathway relevant to Olig2 target genes in motor neuron progenitor cells. Entrez Gene IDs of 5966 Olig2 target genes in pMNs were imported into the Functional Annotation tool of DAVID. It extracted the KEGG "Glioma" pathway (mmu05214) as the 17th rank pathway ([Table 1](#t1-jcnsd-7-2015-001){ref-type="table"}). Olig2 target genes are colored orange.

**Supplementary Figure 4.** KeyMolnet molecular network relevant to Olig2 target genes in motor neuron progenitor cells. Entrez Gene IDs of 5966 Olig2 target genes in pMNs were imported into KeyMolnet. The neighboring network-search algorithm extracted the extremely complex network composed of 5839 molecules and 13,818 molecular relations, showing the most significant relationship with "Transcriptional regulation by p53". Red nodes indicate those closely related to imported genes. White nodes exhibit additional nodes extracted automatically from the core contents of KeyMolnet to establish molecular connections. The molecular relation is indicated by solid line with arrow (direct binding or activation), solid line with arrow and stop (direct inactivation), solid line without arrow (complex formation), dash line with arrow (transcriptional activation), and dash line with arrow and stop (transcriptional repression).

**Supplementary Table 1.** The set of 5,966 ChIP-Seq-based Olig2 target genes in motor neuron progenitor cells.

**Supplementary Table 2.** The set of 1,553 ChIP-Seq-based Olig2 target genes in oligodendrocyte progenitor cells.

**Supplementary Table 3.** The set of 740 Olig2 target genes overlapping bewteen motor neuron progenitor cells and oligodendrocyte progenitor cells.

**Supplementary Table 4.** The highly stringent set of 4,717 ChIP-Seq-based Olig2 target genes in motor neuron progenitor cells.

**Supplementary Table 5.** The set of 277 genes downregu-lated in motor neurons of mutant SOD1 transgenic mice

**Supplementary Table 6.** The set of 1,583 genes down-regulated in lumbar spinal cord tissues of ALS.

The authors thank Ms. Mutsumi Motouri for her invaluable help.

**ACADEMIC EDITOR:** Alexander Rotenberg, Editor in Chief

**FUNDING:** This work was supported by the JSPS KAKENHI (C22500322 and C25430054), the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, the Science Research Promotion Fund of the Promotion and Mutual Aid Corporation for Private Schools of Japan and a grant from the National Center for Geriatrics and Gerontology (NCGG26--20). The authors confirm that the funder had no influence over the study design, content of the article, or selection of this journal.

**COMPETING INTERESTS:** Authors disclose no potential conflicts of interest.

Paper subject to independent expert blind peer review by minimum of two reviewers. All editorial decisions made by independent academic editor. Upon submission manuscript was subject to anti-plagiarism scanning. Prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. This journal is a member of the Committee on Publication Ethics (COPE).

**Author Contributions**

Designed the methods, analyzed the data, and drafted the manuscript: JS. Helped in data analysis: NA, SK, YK. All authors have read and approved the final manuscript.

![Genomic location of Olig2 ChIP-Seq peak on the *Nkx2-2* gene. The genomic location of Olig2 ChIP-Seq peak was determined by importing the processed data into GenomeJack. An example of homeobox protein Nkx2--2 (Nkx2.2; Entrez Gene ID 18088) is shown, where a MACS peak numbered 22463 in the Olig2.bam Coverage track is located in the intronic region of the *Nkx2-2* gene, isoform 2 (panel **A**) with an E-box consensus sequence motif highlighted by orange square (panel **B**).](jcnsd-7-2015-001f1){#f1-jcnsd-7-2015-001}

![KEGG "Axon guidance" pathway relevant to Olig2 target genes in motor neuron progenitor cells. Entrez Gene IDs of 5966 Olig2 target genes in pMNs were imported into the Functional Annotation tool of DAVID. It extracted the KEGG "Axon guidance" pathway (mmu04360) as the second rank pathway ([Table 1](#t1-jcnsd-7-2015-001){ref-type="table"}). Olig2 target genes are colored orange.](jcnsd-7-2015-001f2){#f2-jcnsd-7-2015-001}

![IPA "RNA post-transcriptional modification, embryonic development, hair and skin development and function" network relevant to Olig2 target genes in motor neuron progenitor cells. Entrez Gene IDs of 5966 Olig2 target genes in pMNs were imported into the Core Analysis tool of IPA. It extracted the "RNA post-transcriptional modification, embryonic development, hair and skin development and function" network as the third rank functional network. Olig2 target genes are colored green. Both CUL1 and CUL3 serve as a hub in the network.](jcnsd-7-2015-001f3){#f3-jcnsd-7-2015-001}

######

KEGG pathways relevant to ChIP-Seq-based Olig2 target genes in motor neuron progenitor cells.

  RANK   PATHWAYS                                                           FOCUSED GENES                                                                                *P*-VALUE (BONFERRONI CORRECTED)   FDR
  ------ ------------------------------------------------------------------ ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ---------------------------------- -------------
  1      mmu05200: Pathways in cancer                                       Abl1, Acvr1c, Akt1, Akt3, Apc, Appl1, Arnt2, Axin2, Bax, Bcl2, Bcl2l1, Bcr, Casp3, Casp9, Cblc, Ccdc6, Ccnd1, Cdh1, Cdk2, Cdk4, Cdk6, Cdkn1a, Cdkn2a, Cdkn2b, Cebpa, Chuk, Cks1b, Col4a1, Col4a2, Col4a4, Col4a6, Crkl, Ctbp2, Ctnna1, Ctnna2, Ctnna3, Ctnnb1, Cycs, Dapk1, Dapk2, Dcc, E2f1, E2f3, Egfr, Egln2, Egln3, Erbb2, Fadd, Fas, Fgf1, Fgf10, Fgf11, Fgf12, Fgf14, Fgf18, Fgf2, Fgf21, Fgf7, Fgfr1, Fgfr2, Fgfr3, Flt3, Flt3l, Fn1, Fzd2, Fzd5, Fzd7, Fzd8, Fzd9, Gli1, Gli2, Gli3, Hhip, Hsp90ab1, Hsp90b1, Igf1, Igf1r, Itga2, Itga6, Itgav, Itgb1, Jak1, Jun, Jup, Kit, Kitl, Lama1, Lama3, Lama5, Lamb3, Lamc1, Lef1, Mapk1, Mapk10, Mapk3, Mecom, Met, Mitf, Mlh1, Msh3, Mtor, Nfkbia, Ntrk1, Pdgfrb, Pias2, Pias4, Pik3cb, Pik3r1, Pik3r2, Plcg1, Plcg2, Ppard, Pparg, Prkca, Prkcb, Ptch1, Ptch2, Ptk2, Ralgds, RarA, Rarb, Rb1, Ret, Runx1t1, Rxra, Rxrb, Rxrg, Sfpi1, Slc2a1, Smad2, Smad3, Sos1, Stat1, Stat3, Sufu, Tcf7l1, Tcf7l2, Tgfa, Tgfb2, Tpm3, Traf4, Traf5, Traf6, Trp53, Vegfc, Wnt1, Wnt10a, Wnt5a, Wnt5b, Wnt7a, Wnt8b, Wnt9a, Zbtb16   4.29E-14                           2.78E-13
  2      mmu04360: Axon guidance                                            Abl1, Ablim1, Ablim2, Ablim3, Arhgef12, Cxcr4, Dcc, Dpysl2, Dpysl5, Efna5, Efnb1, Efnb2, Efnb3, Epha1, Epha2, Epha3, Epha4, Epha8, Ephb1, Ephb2, Ephb3, Fyn, Gnai2, Itgb1, Limk1, Mapk1, Mapk3, Met, Nck2, Nfatc1, Nfatc4, Ngef, Nrp1, Ntn1, Ntn4, Ntng1, Pak2, Pak3, Pak4, Pak6, Pak7, Plxna1, Plxna2, Plxna4, Plxnb2, Plxnc1, Ppp3ca, Ptk2, Rgs3, Robo1, Robo2, Robo3, Rock2, Sema3a, Sema3c, Sema3e, Sema3g, Sema4b, Sema5a, Sema5b, Sema6a, Sema6b, Sema6d, Slit1, Slit3, Srgap1, Srgap2, Srgap3, Unc5a, Unc5b, Unc5c, Unc5d                                                                                1.76E-09                           1.14E-08
  3      mmu04510: Focal adhesion                                           Actb, Actn1, Actn3, Actn4, Akt1, Akt3, Bcar1, Bcl2, Capn2, Cav1, Ccnd1, Ccnd2, Ccnd3, Col1a1, Col2a1, Col4a1, Col4a2, Col4a4, Col4a6, Col5a2, Col6a6, Crkl, Ctnnb1, Diap1, Dock1, Egfr, Elk1, Erbb2, Flna, Flnb, Flnc, Flt1, Fn1, Fyn, Igf1, Igf1r, Itga1, Itga2, Itga6, Itga8, Itga9, Itgav, Itgb1, Itgb5, Itgb6, Jun, Kdr, Lama1, Lama3, Lama5, Lamb3, Lamc1, Mapk1, Mapk10, Mapk3, Met, Myl7, Myl9, Mylk, Mylk3, Pak2, Pak3, Pak4, Pak6, Pak7, Parva, Parvb, Pdgfd, Pdgfrb, Pik3cb, Pik3r1, Pik3r2, Ppp1cc, Prkca, Prkcb, Ptk2, Rap1a, Rap1b, Reln, Rock2, Shc2, Sos1, Src, Thbs1, Thbs2, Tln2, Tnc, Tnr, Vav2, Vav3, Vegfc, Vwf                                                                                2.90E-07                           1.87E-06
  4      mmu04010:MAPK signaling pathway                                    Acvr1c, Akt1, Akt3, Arrb1, B230120H23Rik, Bdnf, Cacna1a, Cacna1b, Cacna1c, Cacna1d, Cacna1e, Cacna1g, Cacna1h, Cacna1i, Cacna1s, Cacna2d1, Cacna2d2, Cacna2d3, Cacna2d4, Cacnb2, Cacnb3, Cacng2, Cacng4, Cacng5, Casp3, Cdc25b, Chuk, Crkl, Daxx, Dusp5, Dusp6, Dusp7, Dusp8, Ecsit, Egfr, Elk1, Fas, Fgf1, Fgf10, Fgf11, Fgf12, Fgf14, Fgf18, Fgf2, Fgf21, Fgf7, Fgfr1, Fgfr2, Fgfr3, Fgfr4, Flna, Flnb, Flnc, Gadd45g, Gna12, Gng12, Hspa1a, Hspa1b, Hspa2, Hspa8, Jun, Map2k5, Map2k6, Map3k4, Map3k5, Map3k8, Map4k3, Map4k4, Mapk1, Mapk10, Mapk11, Mapk14, Mapk3, Mapk8ip1, Mapk8ip3, Mapkapk2, Mapkapk3, Mapt, Mecom, Mef2c, Mknk1, Nfatc4, Ntf3, Ntrk1, Ntrk2, Pak2, Pdgfrb, Pla2g1b, Pla2g2c, Pla2g2d, Pla2g5, Ppp3ca, Ppp5c, Prkacb, Prkca, Prkcb, Prkx, Rap1a, Rap1b, Rasgrf2, Rasgrp2, Rasgrp3, Rps6ka2, Rps6ka5, Rras2, Sos1, Srf, Stk3, Stmn1, Tab1, Tab2, Taok3, Tgfb2, Traf6, Trp53                                                                                7.28E-07                           4.70E-06
  5      mmu05412: Arrhythmogenic right ventricular cardiomyopathy (ARVC)   Actb, Actn1, Actn3, Actn4, Atp2a2, Cacna1c, Cacna1d, Cacna1s, Cacna2d1, Cacna2d2, Cacna2d3, Cacna2d4, Cacnb2, Cacnb3, Cacng2, Cacng4, Cacng5, Cdh2, Ctnna1, Ctnna2, Ctnna3, Ctnnb1, Dag1, Dmd, Dsp, Emd, Itga1, Itga2, Itga6, Itga8, Itga9, Itgav, Itgb1, Itgb5, Itgb6, Jup, Lef1, Pkp2, Ryr2, Sgcb, Slc8a1, Tcf7l1, Tcf7l2                                                                                9.49E-06                           6.13E-05
  6      mmu05222:Small cell lung cancer                                    Akt1, Akt3, Bcl2, Bcl2l1, Casp9, Ccnd1, Cdk2, Cdk4, Cdk6, Cdkn2b, Chuk, Cks1b, Col4a1, Col4a2, Col4a4, Col4a6, Cycs, E2f1, E2f3, Fhit, Fn1, Itga2, Itga6, Itgav, Itgb1, Lama1, Lama3, Lama5, Lamb3, Lamc1, Nfkbia, Pias2, Pias4, Pik3cb, Pik3r1, Pik3r2, Ptk2, Rarb, Rb1, Rxra, Rxrb, Rxrg, Traf4, Traf5, Traf6, Trp53                                                                                3.03E-05                           1.95E-04
  7      mmu04520: Adherens junction                                        Actb, Actn1, Actn3, Actn4, Acvr1c, Baiap2, Cdh1, Ctnna1, Ctnna2, Ctnna3, Ctnnb1, Ctnnd1, Egfr, Erbb2, Fgfr1, Fyn, Igf1r, Iqgap1, Lef1, Mapk1, Mapk3, Met, Mllt4, Pard3, Ptprb, Ptprf, Ptprj, Ptprm, Pvrl1, Pvrl2, Pvrl3, Smad2, Smad3, Sorbs1, Src, Tcf7l1, Tcf7l2, Wasf2, Wasf3                                                                                0.00168756                         0.010904142
  8      mmu05223: Non-small cell lung cancer                               Akt1, Akt3, Casp9, Ccnd1, Cdk4, Cdk6, Cdkn2a, E2f1, E2f3, Egfr, Erbb2, Fhit, Foxo3, Mapk1, Mapk3, Pik3cb, Pik3r1, Pik3r2, Plcg1, Plcg2, Prkca, Prkcb, Rarb, Rb1, Rxra, Rxrb, Rxrg, Sos1, Tgfa, Trp53                                                                                0.003433104                        0.0222011
  9      mmu04020: Calcium signaling pathway                                Adcy1, Adcy3, Adcy9, Adora2a, Adra1a, Adra1b, Adra1d, Adrb1, Agtr1a, Atp2a2, Atp2b1, Atp2b2, Atp2b3, Atp2b4, Avpr1a, Cacna1a, Cacna1b, Cacna1c, Cacna1d, Cacna1e, Cacna1g, Cacna1h, Cacna1i, Cacna1s, Calm2, Calm3, Camk2a, Camk2b, Camk2d, Cckar, Chrna7, Ednrb, Egfr, Erbb2, Erbb4, F2r, Gna11, Gna14, Gna15, Gnaq, Gnas, Grm5, Htr2c, Itpr2, Itpr3, Lhcgr, Mylk, Mylk3, Nos1, Nos3, Ntsr1, P2rx1, P2rx4, P2rx7, Pde1a, Pde1b, Pde1c, Pdgfrb, Phka2, Plcb1, Plcb4, Plcd1, Plce1, Plcg1, Plcg2, Ppp3ca, Prkacb, Prkca, Prkcb, Prkx, Ptafr, Ptger3, Ryr1, Ryr2, Ryr3, Slc8a1, Slc8a3, Trhr                                                                                0.005823665                        0.037702607
  10     mmu04916: Melanogenesis                                            a, Adcy1, Adcy3, Adcy5, Adcy9, Calm2, Calm3, Camk2a, Camk2b, Camk2d, Creb1, Creb3l1, Ctnnb1, Dct, Edn1, Ednrb, Fzd2, Fzd5, Fzd7, Fzd8, Fzd9, Gnai2, Gnao1, Gnaq, Gnas, Kit, Kitl, Lef1, Mapk1, Mapk3, Mc1r, Mitf, Plcb1, Plcb4, Prkacb, Prkca, Prkcb, Prkx, Tcf7l1, Tcf7l2, Wnt1, Wnt10a, Wnt5a, Wnt5b, Wnt7a, Wnt8b, Wnt9a                                                                                0.00819137                         0.053090309
  11     mmu04144: Endocytosis                                              Acvr1c, Adrb1, Agap1, Agap3, Ap2a1, Ap2b1, Ap2s1, Arap2, Arfgap2, Arrb1, Asap1, Asap2, Asap3, Cblc, Chmp4c, Cxcr2, Cxcr4, Dab2, Dnajc6, Dnm1, Dnm3, Egfr, Ehd1, Ehd2, Ehd3, Epn1, Epn2, Epn3, Eps15, Erbb4, F2r, Fam125b, Fgfr2, Fgfr3, Fgfr4, Flt1, Git2, H2-K1, Hspa1a, Hspa1b, Hspa2, Hspa8, Igf1r, Iqsec1, Iqsec3, Kdr, Kit, Met, Nedd4, Nedd4l, Ntrk1, Pard3, Pard6g, Pip4k2b, Pip5k1b, Pld1, Prkcz, Psd, Psd3, Psd4, Rab11b, Rab11fip1, Rab11fip4, Rab22a, Rab31, Rab5b, Ret, Sh3gl1, Sh3gl3, Sh3glb1, Sh3kbp1, Smurf1, Src, Stam, Stambp, Traf6, Vps24, Vps25, Vps36, Vps37c, Wwp1                                                                                0.008343602                        0.054080841
  12     mmu05210: Colorectal cancer                                        Acvr1c, Akt1, Akt3, Apc, Appl1, Axin2, Bax, Bcl2, Casp3, Casp9, Ccnd1, Ctnnb1, Cycs, Dcc, Egfr, Fzd2, Fzd5, Fzd7, Fzd8, Fzd9, Igf1r, Jun, Lef1, Mapk1, Mapk10, Mapk3, Met, Mlh1, Msh3, Pdgfrb, Pik3cb, Pik3r1, Pik3r2, Ralgds, Smad2, Smad3, Sos1, Tcf7l1, Tcf7l2, Tgfb2, Trp53                                                                                0.008624979                        0.055912058
  13     mmu04540: Gap junction                                             Adcy1, Adcy3, Adcy5, Adcy9, Adrb1, Csnk1d, Drd2, Egfr, Gna11, Gnai2, Gnaq, Gnas, Grm5, Gucy1b2, Htr2c, Itpr2, Itpr3, lpar1, Map2k5, Mapk1, Mapk3, Pdgfd, Pdgfrb, Plcb1, Plcb4, Prkacb, Prkca, Prkcb, Prkg1, Prkx, Sos1, Src, Tuba1a, Tuba1b, Tubb2a, Tubb2b, Tubb2c, Tubb3, Tubb4, Tubb5, Tubb6                                                                                0.008624979                        0.055912058
  14     mmu05414: Dilated cardiomyopathy                                   Actb, Adcy1, Adcy3, Adcy5, Adcy9, Adrb1, Atp2a2, Cacna1c, Cacna1d, Cacna1s, Cacna2d1, Cacna2d2, Cacna2d3, Cacna2d4, Cacnb2, Cacnb3, Cacng2, Cacng4, Cacng5, Dag1, Dmd, Emd, Gnas, Igf1, Itga1, Itga2, Itga6, Itga8, Itga9, Itgav, Itgb1, Itgb5, Itgb6, Myl3, Prkacb, Prkx, Ryr2, Sgcb, Slc8a1, Tgfb2, Tpm2, Tpm3, Tpm4                                                                                0.009683379                        0.06280453
  15     mmu04012: ErbB signaling pathway                                   Abl1, Abl2, Akt1, Akt3, Camk2a, Camk2b, Camk2d, Cblc, Cdkn1a, Crkl, Egfr, Eif4ebp1, Elk1, Erbb2, Erbb4, Jun, Mapk1, Mapk10, Mapk3, Mtor, Nck2, Nrg1, Nrg2, Pak2, Pak3, Pak4, Pak6, Pak7, Pik3cb, Pik3r1, Pik3r2, Plcg1, Plcg2, Prkca, Prkcb, Ptk2, Rps6kb1, Shc2, Sos1, Src, Tgfa                                                                                0.01193122                         0.077465723
  16     mmu04270: Vascular smooth muscle contraction                       Actg2, Adcy1, Adcy3, Adcy5, Adcy9, Adora2a, Adra1a, Adra1b, Adra1d, Agtr1a, Arhgef12, Avpr1a, Cacna1c, Cacna1d, Cacna1s, Calcrl, Cald1, Calm2, Calm3, Gna11, Gna12, Gnaq, Gnas, Gucy1b2, Itpr2, Itpr3, Kcnma1, Kcnmb1, Kcnmb2, Mapk1, Mapk3, Mrvi1, Myl9, Mylk, Mylk3, Pla2g1b, Pla2g2c, Pla2g2d, Pla2g5, Plcb1, Plcb4, Ppp1cc, Ppp1r14a, Prkacb, Prkca, Prkcb, Prkcd, Prkce, Prkch, Prkg1, Prkx, Ramp3, Rock2                                                                                0.016626811                        0.108192921
  17     mmu05214: Glioma                                                   Akt1, Akt3, Calm2, Calm3, Camk2a, Camk2b, Camk2d, Ccnd1, Cdk4, Cdk6, Cdkn1a, Cdkn2a, E2f1, E2f3, Egfr, Igf1, Igf1r, Mapk1, Mapk3, Mtor, Pdgfrb, Pik3cb, Pik3r1, Pik3r2, Plcg1, Plcg2, Prkca, Prkcb, Rb1, Shc2, Sos1, Tgfa, Trp53                                                                                0.023941745                        0.15633521
  18     mmu04340: Hedgehog signaling pathway                               Bmp6, Bmp7, Bmp8a, Btrc, Csnk1d, Csnk1e, Csnk1g1, Csnk1g2, Csnk1g3, Gas1, Gli1, Gli2, Gli3, Hhip, Lrp2, Prkacb, Prkx, Ptch1, Ptch2, Rab23, Sufu, Wnt1, Wnt10a, Wnt5a, Wnt5b, Wnt7a, Wnt8b, Wnt9a                                                                                0.033019266                        0.216549231
  19     mmu05218: Melanoma                                                 Akt1, Akt3, Ccnd1, Cdh1, Cdk4, Cdk6, Cdkn1a, Cdkn2a, E2f1, E2f3, Egfr, Fgf1, Fgf10, Fgf11, Fgf12, Fgf14, Fgf18, Fgf2, Fgf21, Fgf7, Fgfr1, Igf1, Igf1r, Mapk1, Mapk3, Met, Mitf, Pdgfd, Pdgfrb, Pik3cb, Pik3r1, Pik3r2, Rb1, Trp53                                                                                0.039434735                        0.259424773
  20     mmu04810: Regulation of actin cytoskeleton                         Abi2, Actb, Actn1, Actn3, Actn4, Apc, Arhgef12, Arhgef7, Baiap2, Bcar1, Crkl, Cyfip2, Diap1, Diap3, Dock1, Egfr, Enah, Ezr, F2r, Fgf1, Fgf10, Fgf11, Fgf12, Fgf14, Fgf18, Fgf2, Fgf21, Fgf7, Fgfr1, Fgfr2, Fgfr3, Fgfr4, Fn1, Gna12, Gng12, Iqgap1, Itga1, Itga2, Itga6, Itga8, Itga9, Itgae, Itgam, Itgav, Itgb1, Itgb5, Itgb6, Limk1, Mapk1, Mapk3, Msn, Myh9, Myl7, Myl9, MylK, Mylk3, Nckap1l, Pak2, Pak3, Pak4, Pak6, Pak7, Pdgfd, Pdgfrb, Pik3cb, Pik3r1, Pik3r2, Pip4k2a, Pip4k2b, Pip5k1b, Ppp1cc, Ptk2, Rock2, Rras2, Slc9a1, Sos1, Ssh1, Tiam1, Tiam2, Vav2, Vav3, Wasf2                                                                                0.042152262                        0.277667235
  21     mmu04512: ECM-receptor interaction                                 Agrn, CD44, Col1a1, Col2a1, Col4a1, Col4a2, Col4a4, Col4a6, Col5a2, Col6a6, Dag1, Fn1, Itga1, Itga2, Itga6, Itga8, Itga9, Itgav, Itgb1, Itgb5, Itgb6, Lama1, Lama3, Lama5, Lamb3, Lamc1, Reln, Sdc2, Sdc3, Sdc4, Sv2a, Sv2b, Sv2c, Thbs1, Thbs2, Tnc, Tnr, Vwf                                                                                0.047806047                        0.315776127

**Notes:** By importing Entrez Gene IDs of 5,966 ChIP-Seq-based Olig2 target genes in pMNs into the Functional Annotation tool of DAVID, KEGG pathways showing significant relevance to the set of imported genes were identified. They are listed with rank, pathways, focused genes, *P*-value corrected by Bonferroni multiple comparison test, and false discovery rate (FDR).

######

The set of 77 Olig2 target genes downregulated in motor neurons of mutant SOD1 transgenic mice.

  ENTREZ GENE ID   GENE SYMBOL     GENE NAME                                                                                LOCUS                        LOG2(FOLD CHANGE)   Q-VALUE
  ---------------- --------------- --------------------------------------------------------------------------------------------------- ---------------------------- ------------------- -------------
  319239           Npsr1           neuropeptide S receptor 1                                                                           chr9:23902461--24122130      −3.78453            0.000161548
  214642           A430107O13Rik   RIKEN cDNA A430107O13 gene                                                                          chr6:21935909--22205606      −3.64386            0.00288767
  216974           Proca1          protein interacting with cyclin A1                                                                  chr11:78006893--78019265     −3.18442            0.00113794
  94216            Col4a6          collagen, type IV, alpha 6                                                                          chrX:137599945--137908619    −3.09954            0.000161548
  245038           Dclk3           doublecortin-like kinase 3                                                                          chr9:111341584--111392115    −2.66985            0.000161548
  213980           Fbxw10          F-box and WD-40 domain protein 10                                                                   chr11:62660624--62690964     −2.62803            0.000161548
  14120            Fbp2            fructose bisphosphatase 2                                                                           chr13:62938244--62959730     −2.49476            0.00587005
  227327           B3gnt7          UDP-GlcNAc:betaGal beta-1,3-N- acetylglucosaminyltransferase 7                                      chr1:88199795--88203880      −2.365              0.000161548
  331524           Xkrx            X Kell blood group precursor related X linked                                                       chrX:130683583--130696467    −2.28876            0.000161548
  22095            Tshr            thyroid stimulating hormone receptor                                                                chr12:92236931--92786285     −2.0461             0.000161548
  237759           Col23a1         collagen, type XXIII, alpha 1                                                                       chr11:51103421--51397427     −2.0398             0.000161548
  20394            Scg5            secretogranin V                                                                                chr2:113616469--113669248    −1.97998            0.000161548
  140709           Emid2           EMI domain containing 2                                                                             chr5:137217633--137358977    −1.92523            0.000161548
  228846           D630003M21Rik   RIKEN cDNA D630003M21 gene                                                                          chr2:158008268--158054958    −1.81897            0.000161548
  71907            Serpina9        serine (or cysteine) peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 9   chr12:105234829--105251862   −1.78003            0.00139798
  67313            5730559C18Rik   RIKEN cDNA 5730559C18 gene                                                                          chr1:138110098--138130857    −1.74702            0.000310928
  22409            Wnt10a          wingless related MMTV integration site 10a                                                          chr1:74838592--74850749      −1.74444            0.000161548
  69047            Atp2c2          ATPase, Ca++ transporting, type 2C, member 2                                                        chr8:122223908--122281618    −1.68066            0.000161548
  102626           Mapkapk3        mitogen-activated protein kinase-activated protein kinase 3                                         chr9:107157257--107192208    −1.62955            0.000161548
  18227            Nr4a2           nuclear receptor subfamily 4, group A, member 2                                                     chr2:56959636--56976414      −1.60877            0.000161548
  107371           Exoc6           exocyst complex component 6                                                                         chr19:37611363--37767797     −1.59946            0.00276769
  380787           A230065H16Rik   RIKEN cDNA A230065H16 gene                                                                          chr12:112644994--112650288   −1.54378            0.000161548
  432611           Dnaic2          dynein, axonemal, intermediate chain 2                                                              chr11:114588725--114619200   −1.52146            0.000310928
  67168            Lpar6           purinergic receptor P2Y, G-protein coupled, 5                                                       chr14:73595308--73725598     −1.49476            0.000161548
  13836            Epha2           Eph receptor A2                                                                                chr4:140857135--140885299    −1.45943            0.000161548
  27428            Shroom3         shroom family member 3                                                                              chr5:93112460--93394785      −1.42323            0.000161548
  243911           Kirrel2         kin of IRRE like 2 (Drosophila)                                                                     chr7:31232784--31242534      −1.42158            0.000161548
  320415           Gchfr           GTP cyclohydrolase I feedback regulator                                                             chr2:118993523--118998125    −1.37907            0.000161548
  224008           2310008H04Rik   RIKEN cDNA 2310008H04 gene                                                                          chr16:15887378--16146926     −1.37707            0.000161548
  66922            Rras2           related RAS viral (r-ras) oncogene homolog 2                                                        chr7:121190295--121261295    −1.35527            0.000161548
  23962            Oasl2           2′-5′ oligoadenylate synthetase-like 2                                                              chr5:115346942--115362254    −1.3523             0.000161548
  24001            Tiam2           T-cell lymphoma invasion and metastasis 2                                                           chr17:3326572--3557713       −1.35202            0.000161548
  14403            Gabrd           gamma-aminobutyric acid (GABA) A receptor, subunit delta                                            chr4:154759087--154772178    −1.33198            0.000161548
  72823            Pard3b          par-3 partitioning defective 3 homolog B (C. elegans)                                               chr1:61685397--62688858      −1.31316            0.00126983
  12258            Serping1        serine (or cysteine) peptidase inhibitor, clade G, member 1                                         chr2:84605516--84615586      −1.31077            0.000161548
  170788           Crb1            crumbs homolog 1 (Drosophila)                                                                       chr1:141094830--141273653    −1.28699            0.00100681
  27390            Mmel1           membrane metallo-endopeptidase- like 1                                                              chr4:154230308--154273345    −1.2868             0.000161548
  59010            Sqrdl           sulfide quinone reductase-like (yeast)                                                              chr2:122591094--122635287    −1.28591            0.000161548
  259300           Ehd2            EH-domain containing 2                                                                              chr7:16534335--16552884      −1.28353            0.000161548
  231991           Creb5           cAMP responsive element binding protein 5                                                           chr6:53397542--53645826      −1.27894            0.000161548
  13824            Epb4.1l4a       erythrocyte protein band 4.1-like 4a                                                                chr18:33955980--34166860     −1.26689            0.000161548
  67578            Patl2           protein associated with topoisomerase II homolog 2 (yeast)                                          chr2:121945843--122011925    −1.26105            0.000161548
  212706           N4bp3           NEDD4 binding protein 3                                                                             chr11:51456476--51471183     −1.25585            0.000161548
  320782           Tmem154         transmembrane protein 154                                                                           chr3:84470113--84508497      −1.24413            0.000161548
  238725           Gpr150          G protein-coupled receptor 150                                                                      chr13:76192298--76194444     −1.23333            0.000161548
  14675            Gna14           guanine nucleotide binding protein, alpha 14                                                        chr19:16510156--16685308     −1.23149            0.000161548
  216343           Tph2            tryptophan hydroxylase 2                                                                            chr10:114515696--114622078   −1.23133            0.000161548
  226413           Lct             lactase                                                                                chr1:130181340--130224895    −1.21798            0.000161548
  20148            Dhrs3           dehydrogenase/reductase (SDR family) member 3                                                       chr4:144482729--144517548    −1.20253            0.000161548
  52570            Ccdc69          coiled-coil domain containing 69                                                                    chr11:54863239--54891633     −1.18442            0.000161548
  57915            Tbc1d1          TBC1 domain family, member 1                                                                        chr5:64551449--64742725      −1.17336            0.000161548
  20975            Synj2           synaptojanin 2                                                                                chr17:5941279--6079739       −1.17159            0.000161548
  380918           Siah3           seven in absentia homolog 3 (Drosophila)                                                            chr14:75855781--75927095     −1.16995            0.000161548
  15208            Hes5            hairy and enhancer of split 5 (Drosophila)                                                          chr4:154335031--154336480    −1.15668            0.000161548
  209225           Zfp710          zinc finger protein 710                                                                             chr7:87169699--87237716      −1.15627            0.000161548
  12398            Cbfa2t3         core-binding factor, runt domain, alpha subunit 2, translocated to, 3 (human)                       chr8:125149035--125223009    −1.1289             0.000161548
  232680           Cpa2            carboxypeptidase A2, pancreatic                                                                     chr6:30491641--30514473      −1.12326            0.000161548
  18548            Pcsk1           proprotein convertase subtilisin/kexin type 1                                                       chr13:75227434--75269946     −1.11356            0.000161548
  70484            Slc35d2         solute carrier family 35, member D2                                                                 chr13:64197617--64230638     −1.113              0.000310928
  56485            Slc2a5          solute carrier family 2 (facilitated glucose transporter), member 5                                 chr4:149493432--149518277    −1.10736            0.000161548
  212377           Mms22l          MMS22-like, DNA repair protein                                                                      chr4:24423608--24530095      −1.10128            0.000161548
  74762            Mdga1           MAM domain containing glycosylphosphatidylinositol anchor 1                                         chr17:29964902--30024827     −1.10036            0.000161548
  24115            Best1           bestrophin 1                                                                                chr19:10059661--10076123     −1.09578            0.000161548
  208213           Tmem132c        transmembrane protein 132C                                                                          chr5:127722195--128046160    −1.08022            0.000161548
  320226           4930473A06Rik   RIKEN cDNA 4930473A06 gene                                                                          chr4:83171448--83511168      −1.06949            0.000161548
  68180            Hyi             hydroxypyruvate isomerase homolog (E. coli)                                                         chr4:118032603--118081868    −1.06726            0.000161548
  223864           Rapgef3         Rap guanine nucleotide exchange factor (GEF) 3                                                      chr15:97575200--97598097     −1.06195            0.000161548
  74376            Myo18b          myosin XVIIIb                                                                                chr5:113117895--113325382    −1.06154            0.0030034
  18125            Nos1            nitric oxide synthase 1, neuronal                                                                   chr5:118317127--118406984    −1.03899            0.000161548
  239845           Gpr156          G protein-coupled receptor 156                                                                      chr16:37916581--38007615     −1.03242            0.000161548
  56419            Diap3           diaphanous homolog 3 (Drosophila)                                                                   chr14:87055269--87540987     −1.03241            0.000161548
  18768            Pkib            protein kinase inhibitor beta, cAMP dependent, testis specific                                      chr10:57351786--57460918     −1.03089            0.000161548
  320405           Cadps2          Ca2+-dependent activator protein for secretion 2                                                    chr6:23212773--23789421      −1.0231             0.000161548
  11554            Adrb1           adrenergic receptor, beta 1                                                                         chr19:56796861--56799352     −1.02296            0.00640302
  26903            Dysf            dysferlin                                                                                chr6:83958252--84161039      −1.02138            0.000161548
  11610            Agtrap          angiotensin II, type I receptor- associated protein                                                 chr4:147451169--147462173    −1.01818            0.000161548

**Notes:** By analyzing RNA-Seq data numbered SRP013849, we identified the set of 277 genes significantly downregulated in LCM-purified spinal cord motor neurons of SOD1 G85R transgenic mice, which satisfy Q-value (FDR-adjusted *P*-value) ≤0.01 and log2 fold change (FC) ≤ −1.0 ([Supplementary Table 5](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}). Among them, the set of 77 genes corresponding to Olig2 target genes in pMNs ([Supplementary Table 1](#s1-jcnsd-7-2015-001){ref-type="supplementary-material"}) are listed with Entrez Gene ID, Gene Symbol, Gene Name, Locus, LOG2(FC), and Q-Value.


Introduction
============

Left ventricular (LV) dysfunction is found in about 20--30% of patients with atrial fibrillation (AF). Conversely, AF occurrence can deteriorate LV function determining severe LV dysfunction.[@suz036-B1] Moreover, symptoms of heart failure (HF) like dyspnoea or chest pain may be related to AF occurrence without more specific symptoms like palpitations.[@suz036-B1] Finally, devices like implantable cardioverter-defibrillator (ICD) may register atrial high-rate episodes (AHREs), which can last for more than 5 or 6 min and are also called subclinical AF. The clinical management of these episodes is still controversial. Rhythm vs. rate control strategies is still a matter of debate. Evidences regarding the use of oral anticoagulation in patients with AHRE are scarce. Therefore, AF needs to be confirmed either by surface electrocardiogram (ECG) or by intracardiac electrograms.

Case presentation
=================

A 64-year-old Caucasian man, with history of hypertension and previous subclinical myocardial infarction 10 years before (he was treated with single coronary artery bypass graft: left internal mammalian artery on left anterior descending artery), was admitted to our emergency department for aborted sudden cardiac death related to ventricular fibrillation. A dual chamber ICD (Biotronik Iforia 3 DR-T) was implanted in secondary prevention (*Figure [1](#suz036-F1){ref-type="fig"}*).[@suz036-B2] LV ejection fraction was 44%. The ICD was routinely checked after the implant, and the patient received remote monitoring (RM) system.[@suz036-B3] Five years later, AHRE was detected by RM, and the patient was called for an outpatient visit (*Figure [2](#suz036-F2){ref-type="fig"}*). Atrial fibrillation episode was discovered lasting for more than 24 h and the patient was complaining for occurrence of dyspnoea and chest pain but he was not feeling palpitation or rhythm irregularity. An echocardiogram was repeated and LV ejection fraction felt down to 30% and mild left atrial dilatation was found. The patient was hospitalized for initial HF symptoms and suspect angina. A coronary angiography showed patency of the bypass graft and no critical stenosis on native coronary arteries. The first therapeutic action was to start antithrombotic therapy with rivaroxaban.[@suz036-B1] The thrombo-embolic risk of the patient was classified as high with a CHA~2~DS~2~VASc score of 4. The second action was to choose between rhythm or rate control strategy. Initially a rate control approach was followed, starting treatment with beta-blocker (bisoprolol 10 mg/day). However, after 3 months, patient was still complaining of symptoms, mainly fatigue, dyspnoea, and chest pain during stress. Since the lack of improvement in symptoms, we decided to switch to a rhythm control strategy.[@suz036-B1] Amiodarone therapy was started, and an internal electrical cardioversion from the device was performed restoring sinus rhythm (*Figure [3](#suz036-F3){ref-type="fig"}*).

![Dual chamber implantable cardioverter-defibrillator with atrial and ventricular lead.](suz036f1){#suz036-F1}

![Atrial fibrillation episode detected with remote monitoring. In the upper intracardiac electrogram (EGM) strip sinus rhythm is still present, in the other two EGM strips atrial fibrillation is present since the atrial channel (A) shows much more waves than ventricular channel (V).](suz036f2){#suz036-F2}

![Sinus rhythm was restored: the relationship between atrial (A) and ventricular (V) signals is 1:1.](suz036f3){#suz036-F3}

At 1-month follow-up after electrical cardioversion, the patient was feeling better, no more chest pain, fatigue, or dyspnoea were reported. An echocardiogram performed after 60 days showed an improved LV function with an ejection fraction of 38%. After 3 years of follow-up, AF burden was still 0% and no more AHREs episodes were detected from the ICD, and the patient was in New York Heart Association (NYHA) functional Class I (*Figure [4](#suz036-F4){ref-type="fig"}*).

![Atrial fibrillation burden is showed. In particular, in September 2015 AF burden is 100% until February 2016. Before and after this period atrial fibrillation burden is 0% and in 2018 atrial fibrillation burden is still 0% confirming that the patient is still in sinus rhythm.](suz036f4){#suz036-F4}

Discussion
==========

Atrial fibrillation and HF frequently coexist in the same patient, often negatively promoting the course of each other. Dyspnoea or chest pain without more specific arrhythmic symptoms may delay the diagnosis of AF in HF patients. Given the poor outcomes in patients with HF who developed AF, a prompt diagnosis and therapy could be of paramount importance in these patients.

Cardiac implantable electronic devices (CIEDs) with an atrial lead or dipole for sensing allow continuous monitoring of atrial rhythm potentially highlighting AHRE that enable early detection of AF. Prior studies have demonstrated that the detection of AHRE correlates with electrocardiographic documentation of AF.[@suz036-B4]^,^[@suz036-B5]

Atrial fibrillation can affect HF triggering inappropriate shocks, precipitating HF, and increasing the risk of thrombo-embolic stroke. The principal benefits of RM are early arrhythmia detection associated with a prompt clinical management and patient continuous monitoring allowing patient-tailored strategy of treatment.[@suz036-B6]^,^[@suz036-B7] The Home Guide Registry demonstrated that automatic RM allows early detection of AF with high sensitivity and appropriate reaction to optimize medical treatment.[@suz036-B8] Useful and detailed information about AF episodes such as arrhythmia recurrences and burden, number and duration of the episodes, mean and maximum ventricular rate are stored differently in CIEDs according to the manufacturer.

If the reliability of RM for AF early detection, notification and quantification is well established,[@suz036-B9] evidences regarding the use of this technology for early anticoagulation are less clear.[@suz036-B12] Ongoing clinical trials will probably better clarify the benefit of oral anticoagulation in patients with AHRE \[Apixaban for the Reduction of Thrombo-Embolism in Patients With Device-Detected Sub-clinical Atrial Fibrillation (ARTESIA) and Non-vitamin K antagonist Oral anticoagulations in patients with Atrial High-rate episodes (NOAH--AFNET)\].

In our patient, rhythm control strategy showed better results compared to rate control strategy to control symptoms, in particular chest pain during stress.

Guidelines suggest that HF patients with AF and severe symptoms may benefit from rhythm control therapy in addition to rate control.[@suz036-B1] Several studies and meta-analysis tried to compare rhythm vs. rate control strategy in patients with HF.[@suz036-B16] Although no significant reduction of mortality rate, hospitalization, or thromboembolism was found with rhythm control strategy, persistence of sinus rhythm improves LV function, exercise capacity, and quality of life.[@suz036-B18]^,^[@suz036-B20]^,^[@suz036-B21]

Once the rhythm control strategy was chosen for our patient, the use of a new oral anticoagulant allowed a quicker elective cardioversion compared to vitamin K antagonist (VKA).

In patients undergoing electrical cardioversion, long-term novel oral anticoagulant (NOAC) therapy showed similar results on thrombo-embolic risk prevention compared to warfarin.[@suz036-B22] Moreover, the predictable pharmacokinetics and pharmacodynamics of NOACs allows a more rapid and reliable time to cardioversion without any delay to achieve stable therapeutic international normalized ratio (INR) values as it often the case with VKA strategy.[@suz036-B22]

In conclusion, RM by CIEDs allows early detection of arrhythmia episodes in HF patients even in the absence of clear symptoms. Continuous monitoring enables physician to tailor arrhythmia management and treatment in these patients.

**Conflict of interest**: none declared. The authors didn't receive any financial support in terms of honorarium by Servier for the supplement articles.


Q:

How to add a boolean datatype column to an existing table in sql?

I have a table called person in my database. I want to add another column to the same table and it's a Boolean datatype column. I have tried following queries but it says syntax error near default. I know this is a common and there are lot of answers. I have tried many of them and couldn't figure out to make it work. So please help me.
queries I have tried
ALTER TABLE person add column "AdminApproved" BOOLEAN SET default FALSE;
ALTER TABLE person alter column "AdminApproved" BOOLEAN SET default FALSE;

I have tried without SET key word too.

A:

In SQL SERVER it is BIT, though it allows NULL to be stored
ALTER TABLE person add  [AdminApproved] BIT default 'FALSE';

Also there are other mistakes in your query

When you alter a table to add column no need to mention column keyword in alter statement
For adding default constraint no need to use SET keyword
Default value for a BIT column can be ('TRUE' or '1') / ('FALSE' or 0). TRUE or FALSE needs to mentioned as string not as Identifier

A:

The answer given by Pரதீப் creates a nullable bool, not a bool, which may be fine for you. For example in C# it would create: bool? AdminApprovednot bool AdminApproved.
If you need to create a bool (defaulting to false):
    ALTER TABLE person
    ADD AdminApproved BIT
    DEFAULT 0 NOT NULL;


Neuroprotective effect of ginkgetin in experimental cerebral ischemia/reperfusion via apoptosis inhibition and PI3K/Akt/mTOR signaling pathway activation.
Ginkgetin, extracted from Ginkgo biloba L leaves, has been demonstrated to have potential anti-inflammatory and immune-suppressive properties. But the neuroprotective effect and potential mechanisms of ginkgetin on cerebral ischemia/reperfusion (IR) injury remain unclear. In this research, we studied the neuroprotective effect of ginkgetin in the middle part of the middle cerebral artery occlusion/reperfusion rat model, by analyzing the apoptosis of brain tissues harvested from treatment groups and control groups using the terminal deoxynucleotidyl transferase dUTP nick-end labeling and apoptosis assays. In addition, we detected the association of the neuroprotective effect of ginkgetin with apoptosis inhibition via the activation of the phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) signaling pathway using Western blot analysis. Our results showed that administration of ginkgetin remarkably reduced brain infarction volumes and neurologic deficits; in addition, reducing apoptotic cell numbers, downregulating the levels of cleaved caspase-3 and Bax, and upregulating the level of Bcl-2 in rats subjected to IR injury in a dose-dependent manner. Moreover, high-dose ginkgetin treatment (100 mg/kg) significantly increased the phosphorylations of Akt and mTOR. Blocking of PI3K by LY294002 clearly decreased its antiapoptotic effect and reduced both Akt and mTOR phosphorylation levels. Taken together, these results for the first time suggest that ginkgetin antagonizes cerebral IR-induced injury by inhibiting apoptosis in rats, and this effect was attenuated by the activation of PI3K/Akt/mTOR signaling pathway.


Two-dimensional diversity: screening human cDNA phage display libraries with a random diversity probe for the display cloning of phosphotyrosine binding domains.
A random phosphopeptide probe (bio-pYZZZ) has been used for the isolation and identification of multiple SH2 domains from human cDNA-displaying phage libraries. In addition, on-phage analysis and quantification of binding affinities for these phage-displayed proteins has shown them to be functional domains, retaining the same characteristics as in their native state.


That's another loss, Kenny. There was one yesterday. There will be another one tomorrow. If you don't see it coming, then you're the only one. I'm not sure why this would be surprising to you. Do you watch baseball?

MRKARNO

05-27-2003, 08:55 PM

:firejerry

Mammoo

05-27-2003, 09:40 PM

The more I think about it, I wonder if the Sox would be more quickly improved by a blockbuster trade than a managerial change.

I'm not a big JM fan, it's just that shaking up the clubhouse with a serious maneuver will have a twofold effect of bringing in better player(s) and sending a message that the ones remaining had better snap out of it or they could be next.

Last night four error game was shameful. That kind of performance should not be tolerated by management!

TornLabrum

05-27-2003, 09:45 PM

Originally posted by lowesox
That's another loss, Kenny. There was one yesterday. There will be another one tomorrow. If you don't see it coming, then you're the only one. I'm not sure why this would be surprising to you. Do you watch baseball?

We won't lose once we're on the road and away from all the media pressure!

Oops...we are on the road!

WinningUgly!

05-27-2003, 09:47 PM

Originally posted by Mammoo
The more I think about it, I wonder if the Sox would be more quickly improved by a blockbuster trade than a managerial change.

I'm not a big JM fan, it's just that shaking up the clubhouse with a serious maneuver will have a twofold effect of bringing in better player(s) and sending a message that the ones remaining had better snap out of it or they could be next.

Last night four error game was shameful. That kind of performance should not be tolerated by management!

I'm all for unloading a couple of our big righthanded hitting goofs(Lee/Thomas/Konerko) for some good contact hitters with some speed &/or defense.

duke of dorwood

05-27-2003, 10:13 PM

:KW

We're just glad to be away from the fans and Media conjecture. We'd rather be here where there is SARS

What a jag

lowesox

05-28-2003, 09:08 AM

Originally posted by Mammoo
The more I think about it, I wonder if the Sox would be more quickly improved by a blockbuster trade than a managerial change.

I'm not a big JM fan, it's just that shaking up the clubhouse with a serious maneuver will have a twofold effect of bringing in better player(s) and sending a message that the ones remaining had better snap out of it or they could be next.

Last night four error game was shameful. That kind of performance should not be tolerated by management!

NO! A trade is a terrible, terrible idea. Think about it - who makes the trades? And with so many players on this team at their all-time lowest value Kenny is sure to get slaughtered in a deal. Besides, the only way he can make a reasonably good move is if the Yankees set everything up for him.

SoxxoS

05-28-2003, 10:53 AM

Originally posted by WinningUgly!
I'm all for unloading a couple of our big righthanded hitting goofs(Lee/Thomas/Konerko) for some good contact hitters with some speed &/or defense.

I don't think Konerko can be described as a "big righthanded hitting goof." You got the big, righthanded and goof part right, though.

Irishsox1

05-28-2003, 11:44 AM

My message to Kenny would be:

You suck!
Why don't you quit!
Your in way over your head!
Your a pompous ass!
Take Jerry Manual out with you!


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While you have learned more about the value of the educational programs in the Center for Academic Achievement, it may still be a challenge to explain the benefits of these services. Keep at it! The parent/guardian’s role in the educational experience is as important as the roles of professors, academic advisors, and FGCU staff, your student will need you to regularly help them work through his/her challenges. Additionally, we encourage parent/guardian's to advocate that your student regularly utilize FGCU campus resources to develop personal responsibility for his/her academic experience.

If you need help communicating the benefits of the CAA to your student, we will be happy to help. Please, contact us at caa@fgcu.edu or 239.590.7906.

Further information for parents/guardians can be found at Parent and Family Programs in the Office of New Student Programs at Florida Gulf Coast University.


Does initial staging or tumor histology better identify asymptomatic brain metastases in patients with non-small cell lung cancer?
To determine whether the distribution, staging features, or tumor histology of non-small cell lung cancer (NSCLC) distinguishes neurologically symptomatic from asymptomatic patients initially diagnosed with lung cancer, and to determine whether these factors may predict the presence of brain metastasis. We performed a retrospective review of 809 patients with NSCLC and brain metastases who were treated in our institution between January 1996 and March 2003. Patients who had brain metastasis on initial staging were included. Thoracic computed tomographic scans were reviewed for lung tumor features and staging. Neurological computed tomographic or magnetic resonance image scans were assessed for distribution of brain metastases. Medical records were reviewed for comprehensive staging, tumor histology, and neurological symptoms. Fisher's exact test was used to determine any differences among tumor histology, staging, and imaging features among patients with or without neurological symptoms. Of the 809 patients, 181 had brain metastasis at initial staging. Among these 181 patients, 120 (66%) presented with neurological symptoms (group 1); 61 (34%) patients were asymptomatic (group 2). Patients with adenocarcinoma and large-cell carcinoma had greater odds of brain metastases than patients with squamous cell carcinoma (p = 0.001). There were 106 (58.6%) patients with adenocarcinoma, 32 (17.7%) with large cell carcinoma, and 18 (9.9%) with squamous cell carcinoma. In both groups, most lung cancers were in the right lung with upper lobe dominance. No significant difference in tumor histology or T stage was found between groups, although group 2 was more likely to have a higher N stage. Of the 181 patients with brain metastasis, 60 (33.1%) had N0 disease, 51 (28.2%) had T1 disease, and 23 (19.2%) had no other metastasis. There was no correlation between number/distribution of brain metastases and tumor histology, although patients with disease in the cerebellum or temporal lobes had a greater likelihood of neurological symptoms (odds ratio 3.7). There was no significant difference in tumor histology, staging, or distribution between symptomatic or asymptomatic patients with NSCLC with brain metastases. The odds of brain metastases were greater in those with adenocarcinoma or large-cell carcinoma.


Researchers studying how to improve graduation rates at public colleges and universities have come up with a surprising and counter-intuitive finding: Many students may fail to complete a bachelor's degree not because the work is too hard — but because they're not challenged enough.

It's well known that colleges with the most selective admissions criteria tend to have the highest graduation rates. But even when researchers compared groups of students who had similar academic qualifications, they consistently found that those attending schools with the more demanding academic requirements were more likely to graduate.

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"There is a net effect related to selectivity that is powerful," says Princeton University president emeritus William Bowen, lead author of Crossing the Finish Line: Completing College at America's Public Universities, out today. While everybody does better at stronger schools, "the difference in outcomes … is greater for minorities than for other people," he says.

The book is based on detailed data for 21 flagship public universities and four statewide higher education systems; it explores how the USA might close gaps in college completion rates — a goal embraced by President Obama as a way to increase the nation's economic competitiveness. The research focuses on public higher education, where more than 75% of students enroll, because part of the mission of public institutions is to serve as an engine of opportunity.

The findings underscore age-old advice: Students should enroll in the most selective college that will admit them. But the problem is not that qualified students are being rejected from academically demanding schools. "They never apply in the first place," Bowen says. And the research found that those aiming too low were most likely to be minorities, low-income students and those whose parents never finished college.

The researchers recommend more effective counseling of high school students as they consider their college options. Also, "We need to understand better why students at more selective places graduate in larger numbers," Bowen adds. "We think we know some reasons. One is peer effects. A second is expectations. If you go to a place where the assumption is that everybody is going to graduate … you don't want to be the person who falls behind."

Molly Corbett Broad, president of the American Council on Education, a non-profit group for college presidents, says the research "tells us that there is potential to improve educational attainment (rates) if we step up to the issues that are standing in the way."


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