Research ArticleNeuroscience

Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP

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Sci. Signal.  22 Aug 2017:
Vol. 10, Issue 493, eaal4165
DOI: 10.1126/scisignal.aal4165
  • Fig. 1 LM511 promotes the survival of mDA neurons.

    (A and B) Analysis of the expression of genes encoding laminin subunits in the ventral midbrain (vMidbrain) (A) or the ventral hindbrain (vHindbrain) (B) at E12.5, as assessed by TruSeq RNA sequencing (RNA-seq). RPKM, reads per kilobase of transcript per million mapped reads. Dotted line represents an arbitrary threshold (1.5-fold). (C) Immunohistochemistry of coronal sections through the ventral midbrain at E10.5 and E12.5 for LAMA5 and the mDA neuron marker TH. Ventricular zone, VZ; intermediate zone, IZ; and marginal zone, MZ. Scale bars, 50 um. (D) Immunocytochemistry of E11.5 midbrain cultures assessing the effect of recombinant laminins on differentiation or survival. LMX1A marks the mDA lineage and TH marks mature mDA neurons. Scale bars, 50 μm. (E and F) Quantification of the percentage of LMX1A+ (E) and TH+ (F) cells in the cultures described in (D). (G) Representative images of immunocytochemical staining for activated caspase-3 (aCASP3; green) and TH (red) in E11.5 primary mouse midbrain neuron cultures treated with the neurotoxin, 6-hydroxydopamine (6-OHDA) and either LM111 or LM511. Scale bars, 10 μm. (H) Percentage of TH+ neurons described in (G) that were also aCASP3+. (I) Representative Western blotting analysis of PTEN in SN4741 cells treated for up to 48 hours with LM511. Data are means ± SEM, n = 3 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001, analysis of variance (ANOVA) analysis with Benjamin-Hochberg posttest (B, E, and F) or two-sided t test (H).

  • Fig. 2 LM511 activates YAP via ITGA3B1 in mDA neurons.

    (A to C) qRT-PCR analysis of the effect of laminins on the expression of YAP-target genes in E11.5 murine mDA neuron cultures (A), murine substantia nigra DA cells (SN4741; B) and human mDA neurons differentiated from hNES-SAI2 cells (C). (D) Representative images of immunocytochemistry assessing the subcellular localization of YAP in primary murine TH+ mDA neurons treated with various laminins, as assessed by immunocytochemistry. Arrowheads indicate cytoplasmic staining of YAP. Scale bars, 10 μm. (E) Quantitative analysis of the ratio of nuclear YAP to total YAP in primary murine TH+ mDA neurons in (D). (F) Effect of blocking antibodies to integrin subunits on the LM511-induced expression of YAP target genes in hNES-SAI2 differentiated into mDA neurons, as assessed by qRT-PCR. (G) Western blot analysis of phosphorylated YAP (Ser127) and PTEN in SN4741 cells treated with LM511 and transfected with Itga3 shRNA (left) or Itga6 shRNA (right) (H) Representative images of immunocytochemistry assessing the subcellular localization of YAP in SN4741 cells transfected with Itga3 shRNA and treated with LM511. Scale bars, 10 μm. (I) Expression of three YAP-target genes in experiments described in (H), as assessed by qRT-PCR. Data are means ± SEM, n = 3 independent experiments; **P < 0.01 and ***P < 0.001, ANOVA analysis with Benjamin-Hochberg posttest (A to C and F) or two-sided t test (E and I).

  • Fig. 3 LM511 regulates miR-130a to repress PTEN via YAP in DA neurons.

    (A) Analysis of the ventral midbrain at E11.5 and E13.5 by immunohistochemistry to identify TH+ and YAP+ cells. Arrows indicate YAP cells with very little or no detectable TH. Scale bars, 20 μm. (B and C) Representative Western blots showing PTEN abundance in SN4741 cells after either YAP knockdown with shRNA (B) or YAP overexpression (C). (D) Assessment of the viability of SN4741 cells after YAP knockdown or overexpression. (E) Immunostaining for aCASP3 to examine cell death in primary TH+ neurons after YAP knockdown or overexpression and 6-OHDA treatment. Scale bars, 10 μm. (F) Representative images of immunohistochemistry analysis of active caspase-3 and TH+ in the ventral midbrain of E14.5 mouse embryos electroporated in utero with control or Yap-shRNA at E11.5. Scale bars, 50 μm. (G) Quantification of active caspase-3 in the cells electroporated (GFP+) in (F). (H to K) qRT-PCR analysis of miRNAs predicted to target PTEN in: different embryonic brain regions (H); laminin-treated mouse primary midbrain cultures (I); SN4741 cells after YAP knockdown or overexpression (J); and SN4741 cells treated with LM511, control, or Yap-shRNA (K). (L) Representative Western blot of SN4741 cells stably overexpressing miR-130a. (M) PTEN immunohistochemistry images of the ventral midbrain of E13.5 embryos electroporated in utero with miR-130a–GFP at E11.5. Scale bar, 50 μm. (N) Analysis of the viability of SN4741 overexpressing YAP after treatment with miR-130a inhibitor. Data are means ± SEM, n = 3 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001, ANOVA analysis with Benjamin-Hochberg posttest (D, H to J, and N) or two-sided t test (G and K).

  • Fig. 4 LM511 and YAP improve DA neuron differentiation.

    (A) Effects of LM511, compared to LM111, on the numbers of mDA neurons and their neurite extension as assessed by immunohistochemistry for NR4A2 and TH. Scale bars, 50 μm. (B and C) qRT-PCR analysis of the effects of LM511, compared to LM111, on the expression of specific DA markers in hNES-AF22 cells (B) or hNES-SAI2 cells differentiated into DA neurons (C). (D and E) Immunocytochemistry analysis of the effect of YAP overexpression on the number of TH+ human DA neurons derived from hNES cells and the levels of PITX3 (D) and LMX1A (E) in TH+ cells. Scale bars, 10 μm. (F) Immunohistochemistry for PITX3 in the midbrain basal plate of E13.5 embryos electroporated in utero with YAP-IRES-GFP or GFP at E11.5. Boxes in the upper right are magnified below. Arrowheads point to ectopic PITX3+ cells. Scale bars, 50 μm. (G) Effects of YAP overexpression on the number of TH+ cells and EdU+ cells as assessed by immunohistochemistry in E13.5 embryos electroporated in utero with YAP-IRES-GFP or GFP in the midbrain floor plate at E11.5. Scale bars, 50 μm. (H) Quantitative analysis of TH+ and EdU+ cells in (G). (I) Schematic diagram of the proposed mechanism by which the LM511-ITGA3B1-YAP axis enhances mDA neuron survival (via suppression of PTEN by miR-130a) and differentiation (by up-regulation of mDA neuron transcription factors, such as LMX1A, LMX1B, and PITX3). Data are means ± SEM, n = 3 independent experiments; *P < 0.05, **P < 0.01, and ***P < 0.001, two-sided t test (B, C, and H).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/493/eaal4165/DC1

    Fig. S1. Expression and function of laminins in the developing mouse brain.

    Fig. S2. Expression of YAP target genes and integrins in ventral midbrain tissue and cell lines.

    Fig. S3. Analysis of the YAP–miR-130a–PTEN pathway in ventral midbrain tissue and cell lines.

    Fig. S4. Effects of the LM511-YAP pathway on human NES cells differentiated into mDA neurons.

    Table S1. Antibodies.

    Table S2. qRT-PCR primers.

    Table S3. Oligonucleotides and cloning primers.

  • Supplementary Materials for:

    Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP

    Dawei Zhang, Shanzheng Yang, Enrique M. Toledo, Daniel Gyllborg, Carmen Saltó, J. Carlos Villaescusa, Ernest Arenas*

    *Corresponding author. Email: ernest.arenas{at}ki.se

    This PDF file includes:

    • Fig. S1. Expression and function of laminins in the developing mouse brain.
    • Fig. S2. Expression of YAP target genes and integrins in ventral midbrain tissue and cell lines.
    • Fig. S3. Analysis of the YAP–miR-130a–PTEN pathway in ventral midbrain tissue and cell lines.
    • Fig. S4. Effects of the LM511-YAP pathway on human NES cells differentiated into mDA neurons.
    • Table S1. Antibodies.
    • Table S2. qRT-PCR primers.
    • Table S3. Oligonucleotides and cloning primers.

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    Citation: D. Zhang, S. Yang, E. M. Toledo, D. Gyllborg, C. Saltó, J. Carlos Villaescusa, E. Arenas, Niche-derived laminin-511 promotes midbrain dopaminergic neuron survival and differentiation through YAP. Sci. Signal. 10, eaal4165 (2017).

    © 2017 American Association for the Advancement of Science

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