Siah2 integrates mitogenic and extracellular matrix signals linking neuronal progenitor ciliogenesis with germinal zone occupancy.

Evidence is lacking as to how developing neurons integrate mitogenic signals with microenvironment cues to control proliferation and differentiation. We determine that the Siah2 E3 ubiquitin ligase functions in a coincidence detection circuit linking responses to the Shh mitogen and the extracellular matrix to control cerebellar granule neurons (CGN) GZ occupancy. We show that Shh signaling maintains Siah2 expression in CGN progenitors (GNPs) in a Ras/Mapk-dependent manner. Siah2 supports ciliogenesis in a feed-forward fashion by restraining cilium disassembly. Efforts to identify sources of the Ras/Mapk signaling led us to discover that GNPs respond to laminin, but not vitronectin, in the GZ microenvironment via integrin ß1 receptors, which engages the Ras/Mapk cascade with Shh, and that this niche interaction is essential for promoting GNP ciliogenesis. As GNPs leave the GZ, differentiation is driven by changing extracellular cues that diminish Siah2-activity leading to primary cilia shortening and attenuation of the mitogenic response.

mTORC1-Mediated Inhibition of 4EBP1 Is Essential for Hedgehog Signaling-Driven Translation and Medulloblastoma.

Mechanistic target of rapamycin (MTOR) cooperates with Hedgehog (HH) signaling, but the underlying mechanisms are incompletely understood. Here we provide genetic, biochemical, and pharmacologic evidence that MTOR complex 1 (mTORC1)-dependent translation is a prerequisite for HH signaling. The genetic loss of mTORC1 function inhibited HH signaling-driven growth of the cerebellum and medulloblastoma. Inhibiting translation or mTORC1 blocked HH signaling. Depleting 4EBP1, an mTORC1 target that inhibits translation, alleviated the dependence of HH signaling on mTORC1. Consistent with this, phosphorylated 4EBP1 levels were elevated in HH signaling-driven medulloblastomas in mice and humans. In mice, an mTORC1 inhibitor suppressed medulloblastoma driven by a mutant SMO that is inherently resistant to existing SMO inhibitors, prolonging the survival of the mice. Our study reveals that mTORC1-mediated translation is a key component of HH signaling and an important target for treating medulloblastoma and other cancers driven by HH signaling.

Seven in Absentia E3 Ubiquitin Ligases: Central Regulators of Neural Cell Fate and Neuronal Polarity.

During neural development, neural precursors transition from a proliferative state within their germinal niches to a migratory state as they relocate to their final laminar positions. Transitions across these states are coupled with dynamic alterations in cellular polarity. This key feature can be seen throughout the developing vertebrate brain, in which neural stem cells give rise to multipolar or unpolarized transit-amplifying progenitors. These transit-amplifying progenitors then expand to give rise to mature neuronal lineages that become polarized as they initiate radial migration to their final laminar positions. The conventional understanding of the cellular polarity regulatory program has revolved around signaling cascades and transcriptional networks. In this review, we discuss recent discoveries concerning the role of the Siah2 ubiquitin ligase in initiating neuronal polarity during cerebellar development. Given the unique features of Siah ubiquitin ligases, we highlight some of the key substrates that play important roles in cellular polarity and propose a function for the Siah ubiquitin proteasome pathway in mediating a post-translational regulatory network to control the onset of polarization.

Bidirectional immune tolerance in nonmyeloablative MHC-mismatched BMT for murine ß-thalassemia.

Nonmyeloablative conditioning using total lymphoid irradiation (TLI) and rabbit antithymocyte serum (ATS) (the murine preclinical equivalent of antithymocyte globulin [ATG]) facilitates immune tolerance after bone marrow transplantation (BMT) across major histocompatibility complex (MHC) disparities and may be a useful strategy for nonmalignant disorders. We previously reported that donor effector T-cell function and graft-versus-host disease (GVHD) are regulated via recipient invariant natural killer T-cell (iNKT) interleukin-4-driven expansion of donor Foxp3+ naturally occurring regulatory T cells (Tregs). This occurs via recipient iNKT- and STAT6-dependent expansion of recipient myeloid dendritic cells (MDCs) that induce contact-dependent expansion of donor Treg through PD-1/PD ligand signaling. After TLI/ATS + BMT, Gr-1lowCD11c+ MDCs and Gr-1highCD11cneg myeloid-derived suppressor cells (MDSCs) were enriched in GVHD target organs. We now report that the recovery of both recipient MDSCs (P < .01) and MDCs (P < .01) is significantly increased when the alkylator cyclophosphamide (CTX) is added to TLI/ATS conditioning. In a BALB/c → B6 lethal GVHD model, adoptive transfer of MDSCs from TLI/ATS/CTX-conditioned recipients is associated with significantly improved GVHD colitis and survival (P < .001), conversion of MDSCs to PD ligand-expressing MDCs, and increased donor naturally occurring Treg recovery (P < .01) compared with control treatment. Using BALB/c donors and ß-thalassemic HW-80 recipients, we found significantly improved rates of engraftment and GVHD following TLI/ATS/CTX compared with TLI/ATS, lethal or sublethal total body irradiation/ATS/CTX, or CTX/ATS conditioning. These data provide preclinical support for trials of TLI/ATG/alkylator regimens for MHC-mismatched BMT for hemoglobinopathies. The data also delineate innate immune mechanisms by which TLI/ATS/CTX conditioning may augment transplantation tolerance.

Zeb1 controls neuron differentiation and germinal zone exit by a mesenchymal-epithelial-like transition.

In the developing mammalian brain, differentiating neurons mature morphologically via neuronal polarity programs. Despite discovery of polarity pathways acting concurrently with differentiation, it's unclear how neurons traverse complex polarity transitions or how neuronal progenitors delay polarization during development. We report that zinc finger and homeobox transcription factor-1 (Zeb1), a master regulator of epithelial polarity, controls neuronal differentiation by transcriptionally repressing polarity genes in neuronal progenitors. Necessity-sufficiency testing and functional target screening in cerebellar granule neuron progenitors (GNPs) reveal that Zeb1 inhibits polarization and retains progenitors in their germinal zone (GZ). Zeb1 expression is elevated in the Sonic Hedgehog (SHH) medulloblastoma subgroup originating from GNPs with persistent SHH activation. Restored polarity signaling promotes differentiation and rescues GZ exit, suggesting a model for future differentiative therapies. These results reveal unexpected parallels between neuronal differentiation and mesenchymal-to-epithelial transition and suggest that active polarity inhibition contributes to altered GZ exit in pediatric brain cancers.

Recipient myeloid-derived immunomodulatory cells induce PD-1 ligand-dependent donor CD4+Foxp3+ regulatory T cell proliferation and donor-recipient immune tolerance after murine nonmyeloablative bone marrow transplantation.

We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4-dependent expansion of donor Foxp3(+) naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2(d)) hosts were administered TLI/ATS and BMT from WT or STAT6(-/-) C57BL/6 (H-2(b)) donors. Following STAT6(-/-) BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4-responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b(+) cell subsets (Gr-1(high)CD11c(-), Gr-1(int)CD11c(-), and Gr-1(low)CD11c(+)) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1(low)CD11c(+) cells induced potent H-2K(b+)CD4(+)Foxp3(+) nTreg proliferation in vitro in 72-h MLRs. Gr-1(low)CD11c(+) cells were reduced significantly in STAT6(-/-) and iNKT cell-deficient Jα18(-/-) BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b(+) cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1(low)CD11c(+) cells to Jα18(-/-) BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3(+) nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-ß signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1(low)CD11c(+) cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand-dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.