The Transcriptional Activator Krüppel-like Factor-6 Is Required for CNS Myelination

Growth factors of the gp130 family promote oligodendrocyte differentiation, and viability, and myelination, but their mechanisms of action are incompletely understood. Here, we show that these effects are coordinated, in part, by the transcriptional activator Krüppel-like factor-6 (Klf6). Klf6 is rapidly induced in oligodendrocyte progenitors (OLP) by gp130 factors, and promotes differentiation. Conversely, in mice with lineage-selective Klf6 inactivation, OLP undergo maturation arrest followed by apoptosis, and CNS myelination fails. Overlapping transcriptional and chromatin occupancy analyses place Klf6 at the nexus of a novel gp130-Klf-importin axis, which promotes differentiation and viability in part via control of nuclear trafficking. Klf6 acts as a gp130-sensitive transactivator of the nuclear import factor importin-α5 (Impα5), and interfering with this mechanism interrupts step-wise differentiation. Underscoring the significance of this axis in vivo, mice with conditional inactivation of gp130 signaling display defective Klf6 and Impα5 expression, OLP maturation arrest and apoptosis, and failure of CNS myelination

The impacts of exercise on age-related cognitive decline.

<p>Three major factors have been implicated in promoting age-related cognitive decline: inflammation, neurovascular changes, and changes in CNS structure and function. Exercise has been shown to be beneficial in impacting these three categories. It has been shown to promote neurogenesis [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002300#pbio.1002300.ref016" target="_blank">16</a>], increase CBF and angiogenesis [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002300#pbio.1002300.ref014" target="_blank">14</a>], and reduce inflammation [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002300#pbio.1002300.ref004" target="_blank">4</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002300#pbio.1002300.ref017" target="_blank">17</a>], correlating with improved cognitive performance. Soto et al. adds to this data (red text) by demonstrating that exercise reduces the age-related loss of pericytes, basement membrane components, and astrocyte reactivity at the NVU, and reduces the amount of complement induction in myeloid cells. <i>Image credit</i>: <i>Gareth R</i>. <i>John & Benjamin M</i>. <i>Laitman</i>.</p

Schematic of the NVU.

<p>The NVU comprises the cerebral microvascular endothelium (shown in red), its basement membrane, and associated pericytes (yellow) and astrocytes (orange). The perivascular space exists between the endothelium and astrocytic endfeet. The endothelium provides the structural and functional basis for the blood–brain barrier (BBB), while astrocytes and pericytes control barrier induction and maintenance [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002300#pbio.1002300.ref007" target="_blank">7</a>]. Junctional proteins exist between endothelial cells and astrocytes (glia limitans) to help regulate entrance into the CNS parenchyma. <i>Image credit</i>: <i>Gareth R</i>. <i>John & Benjamin M</i>. <i>Laitman</i>.</p

Karyopherin Alpha Proteins Regulate Oligodendrocyte Differentiation

<div><p>Proper regulation of the coordinated transcriptional program that drives oligodendrocyte (OL) differentiation is essential for central nervous system myelin formation and repair. Nuclear import, mediated in part by a group of karyopherin alpha (Kpna) proteins, regulates transcription factor access to the genome. Understanding how canonical nuclear import functions to control genomic access in OL differentiation may aid in the creation of novel therapeutics to stimulate myelination and remyelination. Here, we show that members of the Kpna family regulate OL differentiation, and may play distinct roles downstream of different pro-myelinating stimuli. Multiple family members are expressed in OLs, and their pharmacologic inactivation dose-dependently decreases the rate of differentiation. Additionally, upon differentiation, the three major Kpna subtypes (P/α2, Q/α3, S/α1) display differential responses to the pro-myelinating cues T3 and CNTF. Most notably, the Q/α3 karyopherin <i>Kpna4</i> is strongly upregulated by CNTF treatment both compared with T3 treatment and other Kpna responses. <i>Kpna4</i> inactivation results in inhibition of CNTF-induced OL differentiation, in the absence of changes in proliferation or viability. Collectively, these findings suggest that canonical nuclear import is an integral component of OL differentiation, and that specific Kpnas may serve vital and distinct functions downstream of different pro-myelinating cues.</p></div

Karyopherin alphas are widely expression in OLP and important regulators of OL differentiation.

<p><b>(A)</b><i>Kpna</i> gene expression in OLP exposed only to proliferative factors (PDGFAA, 10 ng/ml and bFGF, 20 ng/ml) for 24h. Expression was quantified from isolated total RNA with NanoString nCounter Gene Expression Assay. A panel of probes was designed for target genes and normalized to housekeeping genes <i>Alas1</i>, <i>Ppia</i>, <i>Gapdh</i>, <i>Actb</i>, and <i>Rps11</i>. Following assay completion, raw data was normalized and analyzed using nSolverTM software. <i>Kpna2</i> is the highest expressing Impα in OLP. <i>Kpna7</i> is not expressed in oligodendrocytes. <b>(B-D)</b> Results from primary OLP pre-treated 1h with increasing concentrations the karyopherin inhibitor importazole (IPZ) or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for 9h and harvested for qPCR <b>(B)</b> or immunocytochemistry <b>(C)</b>. <b>(B)</b> Failure of karyopherin functioning in IPZ-treated cultures results in dose-dependent inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers <i>CNP</i> or <i>MBP</i> in qPCR. <b>(C,D)</b> While, at lower doses of IPZ, inhibition of differentiation is seen in the absence of any impact on viability, higher concentrations of IPZ eventually produce apoptosis, quantified as the percentage of DAPI cells that are cleaved-caspse 3 (CC3) positive. <b>(E-I)</b> Extension of differentiation out to 72h with T3 in the presence of 2μM IPZ confirms this reduction in maturation. Results are from primary OLP pre-treated 1h with 2μM IPZ or DMSO, then differentiated with T3 (40ng/ml) in the presence of IPZ/DMSO for up to 72h and harvested for qPCR <b>(E,F)</b> or immunocytochemistry <b>(G-I)</b>. <b>(E,F)</b> Failure of karyopherin functioning in 2μM IPZ-treated cultures results in inhibition of differentiation as measured by a decrease in the expression of the OL maturation markers <i>CNP</i> or <i>MBP</i> in qPCR. <b>(G)</b> Representative image shows maturation markers for immature/mature OL (O4), and mature OL (MBP) in the OL lineage in IPZ-treated cultures and DMSO controls treated with T3 for 72h. Maturation was assessed by quantifying %O4/DAPI <b>(H)</b> and %MBP/DAPI <b>(I)</b> expressing cells. Data presented are mean ± S.E.M. and representative of 3 <b>(A,B,C,E,F)</b> or 5 <b>(H,I)</b> independent cultures. Statistics, <b>(B)</b> One-way ANOVA plus Bonferroni post-test, <b>(D,E,F)</b> Two-way ANOVA plus Bonferroni post-test, <b>(H,I)</b> Student’s t-test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Scale bars, <b>(C,G)</b> 20μm. Individual data values are in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170477#pone.0170477.s001" target="_blank">S1 Data</a></b>.</p

Karyopherin alphas in canonical nuclear import.

<p><b>(A)</b> In canonical import, a karyopherin α (Kpna) binds to both the nuclear localization sequence-containing cargo protein and to Kpnb. Subsequently, Kpnb interacts with the nuclear pore to bring the trimeric complex into the nucleus. Once within the nucleus, the complex dissociates, and Kpna and Kpnb are recycled to the cytoplasm. <b>(B)</b> Seven <i>Kpna</i> genes encoding Kpna proteins have been identified in humans, and six in mice. They belong to three subtypes based on homology. Nomenclature for each corresponding Impα protein is indicated. While nomenclature of the paralogs varies in different studies, we will use the human Kpna designations for clarity.</p

<i>Kpna4</i> is an important regulator of CNTF-induced oligodendrocyte differentiation.

<p><b>(A-C)</b> Confocal imaging of Kpna4 expression <i>in vivo</i> in P1 <b>(A)</b> and P14 <b>(C)</b> thoracolumbar mouse spinal cord sections. Kpna4 is expressed in multiple cells within the CNS including APC⁺Olig2⁺ OLs (representative cells indicated with white arrows), but not by immature Olig2⁺APC^- cells (yellow arrowhead). The region outlined in <b>(A)</b> is magnified in panels to the right. Panel <b>(B)</b> shows a high power image of Olig2⁺ cells in thoracolumbar spinal cord at P1. Cells show both nuclear (<b>B,</b> red arrowheads) and cytoplasmic Kpna4 immunoreactivity (<b>B,</b> white arrowheads). <b>(D)</b> qPCR data from OLP nucleofected with <i>siKpna4</i> or <i>siNT</i> control for 24h. Silencing is efficient and is selective for <i>Kpna4</i>. <b>(E,F)</b> Representative confocal image and associated quantification from primary mouse OLP silenced for <i>Kpna4</i> versus nontargeting control, then allowed to proliferate in the presence of the mitogens PDGFAA and bFGF for 24h. Active proliferation was assessed by immunocytochemistry for Ki67, confocal imaging, and quantitation of %Ki67/DAPI cells. <b>(G-I)</b> To directly compare responses of Kpna4-deficient cells and controls to CNTF vs. T3 in parallel, primary cultures were nucleofected with siRNA for <i>Kpna4</i> or nontargeting (NT) control, then treated with either CNTF or T3. <b>(G)</b> Representative confocal image and associated quantification from primary mouse OLP nucleofected with <i>siKpna4</i> or <i>siNT</i> control and then differentiated with T3 (60 ng/ml) or CNTF (100 ng/ml) for 72h. This image shows maturation markers for OLP (NG2), immature/mature OL (O4), and mature OL (MBP) in the Olig2⁺ OL lineage in <i>Kpna4</i>-deficient cultures and controls treated with T3 or CNTF. Maturation was assessed by quantifying %O4/DAPI <b>(H)</b> and %MBP/DAPI <b>(I)</b> expressing cells. Maturation was reduced in CNTF-treated cultures. In T3-treated cultures, only a slight reduction was observed in the proportion of MBP expressing cells, which did not reach significance, illustrating a differential impact of silencing <i>Kpna4</i> depending on the growth factor added to induce differentiation. <b>(J,K)</b> The reduction in maturation markers coincided with a loss in complexity of branching morphology, a marker of OL maturity, as assessed by fractal analysis. Fractal results are represented by the box-counting fractal dimension (Db). <b>(L,M)</b> Cell death was unchanged in these cultures regardless of treatment with CNTF <b>(L)</b> or T3 <b>(M)</b>, measured by both assessments of apoptosis (%Cleaved-caspase 3 (CC3)/DAPI) and total cell number (DAPI counts per field). Data are mean ± S.E.M. and representative of 3 <b>(D,F,L,M)</b> or 5 <b>(H-K)</b> independent cultures. Statistics, <b>(D,F,L,M,J,K)</b> Student’s t-test, <b>(H,I</b>) Two-way ANOVA plus Bonferroni test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001. Scalebars, <b>(A)</b> 50μm, 25μm inset, <b>(B)</b> 10μM, <b>(C)</b> 25μm, <b>(E)</b> 20μm, <b>(G)</b> 20μm. Individual data values are in <b>S1 data</b>.</p

Oligodendrocyte Kpna expression alters with differentiation.

<p><b>(A,B)</b> T3 and CNTF both induce differentiation of OL. <b>(A)</b> Confocal images of mature OL treated with either T3 (60ng/ml) or CNTF (100ng/ml) for 72h. Cells were immunostained for the earlier maturation marker, O4 (green), and the later myelin protein, MBP (red), a marker of mature OL. <b>(B)</b> Results from qPCR of fold change expression for the maturation markers <i>CNP</i> and <i>MBP</i> at 24h following T3 (left) or CNTF (right) treatment, compared to 0h. <b>(C-H)</b> <i>Kpna</i> gene expression data from OL lineage for 72h treatment with either T3 or CNTF, analyzed at 24h intervals. Transcripts were quantified from isolated total RNA using NanoString nCounter Gene Expression Assay. A commercially available panel of probes for target genes was normalized to housekeeping genes <i>Alas1</i>, <i>Ppia</i>, <i>Gapdh</i>, <i>Actb</i>, and <i>Rps11</i>. Following assay completion, raw data was analyzed using nSolver software before being subjected to statistical analysis. <b>(C-F)</b> Expression fold change in response to T3 (60ng/ml) or CNTF (100ng/ml) at 24h intervals, derived from NanoString analysis. Results demonstrate differential changes in expression of Kpna isotypes during differentiation. <i>Kpna2</i> expression strongly decreases, whereas all other isoforms increase. The three Kpna subtypes (P/α2, Q/α3 and S/α1) also respond differently to the extrinsic factor used. While <i>Kpna2</i> (Subtype P/α2) decreases no matter the cue, <i>Kpna4</i> (Subtype Q/α3) shows greater changes in expression in response to CNTF, and <i>Kpna1</i> and <i>Kpna6</i> (Subtype S/α1) display greater fold changes in response to T3 than other subtypes, in addition to responsiveness to CNTF. <b>(G)</b> Expression fold change derived from NanoString analysis shows that <i>Kpnb1</i> displays relatively stable expression throughout the course of OL differentiation. <b>(H)</b> Expression fold change derived from NanoString analysis demonstrates differential responses of <i>Kpna4</i> to CNTF versus T3 treatment. While expression increases slightly in response to T3, the response to CNTF is greater at all time points beyond 0h. <b>(I,J)</b> Gene expression changes resulted in corresponding alterations in protein levels in response to CNTF. OLP treated for up to 72h with CNTF (100ng/ml) were harvested and immunoblotted for Kpna1, Kpna2, and Kpna4, with Actin used as a loading control <b>(I)</b>. <b>(J)</b> Accompanying densitometry plots for Kpna4 were calculated from the ratio of Kpna4/Actin pixel intensity and displayed as fold change from 0h of CNTF treatment. Data are mean ± S.E.M. and representative of 3 independent cultures. Statistics, <b>(B)</b> Student’s t-test, <b>(C-H)</b> Two-way ANOVA plus Bonferroni post-test, <b>(J)</b> One-way ANOVA plus Bonferroni post-test, *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0001. Statistics for <b>(E-F)</b> are in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170477#pone.0170477.s003" target="_blank">S2 Table</a></b>. Scalebar: <b>(A)</b> 20μm. Individual data values for <b>(C-H)</b> are in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170477#pone.0170477.s001" target="_blank">S1 Table</a></b> and <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170477#pone.0170477.s001" target="_blank">S1 Data</a></b>. Individual data values for <b>(B,J)</b> are in <b>S1 data</b>.</p

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an astrocyte-derived permeability factor, and suggest TYMP and VEGFA together promote blood-brain barrier breakdown