Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

SummaryLamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6α localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement. Ectopic expression or silencing of Par6α inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to “pull” the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6α

Oncogene-dependent apoptosis is mediated by caspase-9

Understanding how oncogenic transformation sensitizes cells to apoptosis may provide a strategy to kill tumor cells selectively. We previously developed a cell-free system that recapitulates oncogene dependent apoptosis as reflected by activation of caspases, the core of the apoptotic machinery. Here, we show that this activation requires a previously identified apoptosis-promoting complex consisting of caspase-9, APAF-1, and cytochrome c. As predicted by the in vitro system, preventing caspase-9 activation blocked drug-induced apoptosis in cells sensitized by E1A, an adenoviral oncogene. Oncogenes, such as E1A, appear to facilitate caspase-9 activation by several mechanisms, including the control of cytochrome c release from the mitochondria

Role of Tet1/3 Genes and Chromatin Remodeling Genes in Cerebellar Circuit Formation.

Although mechanisms underlying early steps in cerebellar development are known, evidence is lacking on genetic and epigenetic changes during the establishment of the synaptic circuitry. Using metagene analysis, we report pivotal changes in multiple reactomes of epigenetic pathway genes in cerebellar granule cells (GCs) during circuit formation. During this stage, Tet genes are upregulated and vitamin C activation of Tet enzymes increases the levels of 5-hydroxymethylcytosine (5hmC) at exon start sites of upregulated genes, notably axon guidance genes and ion channel genes. Knockdown of Tet1 and Tet3 by RNAi in ex vivo cerebellar slice cultures inhibits dendritic arborization of developing GCs, a critical step in circuit formation. These findings demonstrate a role for Tet genes and chromatin remodeling genes in the formation of cerebellar circuitry

Cdc42 Regulates Neuronal Polarity during Cerebellar Axon Formation and Glial-Guided Migration

Summary CNS cortical histogenesis depends on polarity signaling pathways that regulate cell adhesion and motility. Here we report that conditional deletion of the Rho GTPase Cdc42 in cerebellar granule cell precursors (GCPs) results in abnormalities in cerebellar foliation revealed by iDISCO clearing methodology, a loss of columnar organization of proliferating GCPs in the external germinal layer (EGL), disordered parallel fiber organization in the molecular layer (ML), and a failure to extend a leading process and form a neuron-glial junction during migration along Bergmann glia (BG). Notably, GCPs lacking Cdc42 had a multi-polar morphology and slowed migration rate. In addition, secondary defects occurred in BG development and organization, especially in the lateral cerebellar hemispheres. By phosphoproteomic analysis, affected Cdc42 targets included regulators of the cytoskeleton, cell adhesion and polarity. Thus, Cdc42 signaling pathways are critical regulators of GCP polarity and the formation of neuron-glial junctions during cerebellar development

WNT3 activates the MAPK/ERK1,2 and ERK5 signaling pathway(s) in GCPs.

<p>(<i>A</i>,<i>B</i>) GCPs were treated with WNT3 for 24 h and lysates analyzed by Western blot analysis. (<i>A</i>) WNT3 increases ERK1/2 and ERK5 phosphorylation in GCPs. Protein extracts were analyzed by immunoblotting with anti-P-ERK1/2, anti-ERK1/2, anti-P-ERK5, and anti-ERK5 antibodies (n=3). GAPDH was used as loading control. (<i>B</i>) Immunoblotting using anti-P-p38, anti-p38, anti-P-JNK and anti-JNK antibodies showed that WNT3 did not alter p38 or JNK activity. GAPDH was used as loading control. (<i>C</i>) Test of WNT3 specificity using a SRE Luciferase assay in GCPs. BDNF was used as a positive control regulator of MAPK signaling (n=3). (<i>D</i>) MAPK activity induced by WNT3 in GCPs is inhibited by the MEK inhibitor PD98059. SRE-luciferase assay in GCPs treated with control or WNT3, in the absence or presence of PD98059 (n=3). (<i>E</i>). The MEK inhibitor PD98059 reverses WNT3 inhibition of GCP proliferation. As measured by [³H]-Thymidine incorporation, PD98059 increases GCP proliferation by 15%±7%, WNT3 inhibits GCPs proliferation by 34±12% and PD98059 + WNT3 inhibits GCP proliferation by 7%±5% of the control (Con) (n=3). Data represent the mean ± s.e.m.: *p<0.05, **p<0.01, ***p<0.001. NS, not significant.</p

WNT3 decreases the GCP proliferation marker <i>Atoh1</i> and increases the GCP differentiation marker PAX6.

<p>(<i>A</i>) WNT3 decreased <i>Atoh1</i> mRNA levels after 6 h of treatment (Con=1; WNT3: <i>Atoh1</i>=0.52±0.06). (<i>B</i>) Treatment of GCPs with WNT3 decreased <i>Atoh1</i> mRNA levels in the presence of SHH. SHH was added for 24 h and WNT3 was added for 6h. (SHH/Con=1; SHH/WNT3: <i>Atoh1</i>=0.47±0.04). (<i>C</i>) Overexpression of ATOH1 by retroviral infection of GCPs abrogated the WNT3 effect as measured by [³H]-Thymidine incorporation assay. GCPs were infected with retroviruses produced using pMSCV-GFP and pMSCV-<i>Atoh1-GFP</i> on day 1. WNT3 was added to the GCPs on day 2. (GFP/Con, 100±2.5%; GFP/WNT3, 77.4±3.3%; ATOH1-GFP/Con, 98.9±2.3%; ATOH1-GFP/WNT3, 92.6±3.8% (n=3).) (<i>D</i>) WNT3 decreased the mRNA levels of additional mitotic markers Ki67 and <i>Notch2</i> after 24 h of treatment (Con=1; WNT3: <i>Atoh1</i>=0.71±0.07, Ki67=0.70±0.08, and <i>Notch2</i>=0.70±0.06). (<i>E</i>) P7 cerebellar slices were incubated with WNT3 for 24 h, and cryostat sections were immunostained with anti-PAX6 antibody, a marker for GCP differentiation. In control cultures (top panel), low levels of immunostaining are seen with anti-PAX6 antibody. WNT3 treatment (lower panel) increases the intensity of anti-PAX6 immunostaining in GCPs in the external granule layer (EGL), in postmitotic GCPs migrating across the molecular layer (ML, arrow) and in granule neurons in the internal granular layer (IGL) undergoing terminal differentiation. (<i>F</i>) WNT3 increased the mRNA levels of additional post-mitotic markers Zic2 and <i>Gabra6</i> after 24 h of treatment (Con=1; WNT3: Zic2=3.22±0.54 and <i>Gabra6</i>=24.40±2.08). Data represent the mean ± s.e.m.: *p<0.05, **p<0.01, ***p<0.001. NS, not significant. Scale bar 100 µm.</p

GCP proliferation induced by WNT3 is not dependent on the BMP pathway.

<p>(<i>A</i>) WNT3 and BMPs (BMP2, BMP4, BMP6, and BMP7) cooperated to decrease proliferation of GCPs as measured by [³H]-Thymidine incorporation assay. (<i>B</i>) The BMP inhibitor Noggin did not affect the WNT3-mediated decrease in proliferation. (WNT3, 59.9±4.6% of the control value; Con/Noggin, 93.5±1.7%; and WNT3/Noggin 46.4±2.9% (n=3).) (<i>C</i>) GCPs were stimulated for 4h at 1 DIV with WNT3 or BMP7 and analyzed by immunoblotting with the anti-phospho-SMAD1/5/8 antibody. The anti-SMAD1 antibody was used for normalization to obtain the percentage of phosphorylated SMAD1. (Con, 100±8.3%; WNT3, 133.8±33.8%; Con, 100±11.5%; and BMP7, 373.5±88.9% (n=3). Data represent the mean ± s.e.m.: *p<0.05, **p<0.01, ***p<0.001. NS, not significant.</p

WNT3 signaling is not mediated by β-catenin signaling.

<p>(<i>A</i>, <i>B</i>,) GCPs were treated with WNT3 for 24 h and lysates analyzed by Western blot analysis. (<i>A</i>) Immunoblotting of GCP lysates with anti-active-β-catenin (Ac-β-cat) antibody showed that WNT3 did not induce β-catenin activation. The GSK-3 inhibitor BIO was used as positive control to increase activated β-catenin and activated β-catenin levels were compared to total β-catenin levels, using anti-β-catenin antibody (n=3). (<i>B</i>) Immunoblotting using anti-Phospho(P)-216-GSK-3β, anti-GSK-3β and anti-MYCN antibodies showed that WNT3 does not regulate β-catenin signaling. GAPDH was used as loading control. (<i>C</i>) Using a Luciferase assay, WNT3 fails to activate β-catenin signaling, which is reported by the expression of pTOPflash luciferase driven by the TCF/LEF promoter. LiCl was used as a positive control to activate β-catenin signaling. Data represent the mean ± s.e.m.: *p<0.05, **p<0.01, ***p<0.001. NS, not significant.</p

Gene expression profile of Wnts in cerebellar development.

<p>(<i>A</i>) qPCR of Wnt3, Wnt4, Wnt5a, Wnt5b, Wnt7a, and Wnt10b, in the mouse cerebellum from postnatal day 5 (P5) to adult. Wnt levels were normalized against β-2-microglobulin (B2M), hyporanthine-guanine phosphoribosyltransferase (HPRT1) and ribosomal subunit 18s (M18s). (<i>B</i>) WNT3 protein levels during cerebellar development. Immunoblotting of cerebellar lysates from P0 to P56 (adult) with an anti-WNT3 antibody showed an increase in WNT3 protein during cerebellar development. </p