Multiple Nf1 Schwann cell populations reprogram the plexiform neurofibroma tumor microenvironment.

To define alterations early in tumor formation, we studied nerve tumors in neurofibromatosis 1 (NF1), a tumor predisposition syndrome. Affected individuals develop neurofibromas, benign tumors driven by NF1 loss in Schwann cells (SCs). By comparing normal nerve cells to plexiform neurofibroma (PN) cells using single-cell and bulk RNA sequencing, we identified changes in 5 SC populations, including a de novo SC progenitor-like (SCP-like) population. Long after Nf1 loss, SC populations developed PN-specific expression of Dcn, Postn, and Cd74, with sustained expression of the injury response gene Postn and showed dramatic expansion of immune and stromal cell populations; in corresponding human PNs, the immune and stromal cells comprised 90% of cells. Comparisons between injury-related and tumor monocytes/macrophages support early monocyte recruitment and aberrant macrophage differentiation. Cross-species analysis verified each SC population and unique conserved patterns of predicted cell-cell communication in each SC population. This analysis identified PROS1-AXL, FGF-FGFR, and MIF-CD74 and its effector pathway NF-κB as deregulated in NF1 SC populations, including SCP-like cells predicted to influence other types of SCs, stromal cells, and/or immune cells in mouse and human. These findings highlight remarkable changes in multiple types of SCs and identify therapeutic targets for PN.

Cdkn2a Loss in a Model of Neurofibroma Demonstrates Stepwise Tumor Progression to Atypical Neurofibroma and MPNST.

Plexiform neurofibromas are benign nerve sheath Schwann cell tumors characterized by biallelic mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene. Atypical neurofibromas show additional frequent loss of CDKN2A/Ink4a/Arf and may be precursor lesions of aggressive malignant peripheral nerve sheath tumors (MPNST). Here we combined loss of Nf1 in developing Schwann cells with global Ink4a/Arf loss and identified paraspinal plexiform neurofibromas and atypical neurofibromas. Upon transplantation, atypical neurofibromas generated genetically engineered mice (GEM)-PNST similar to human MPNST, and tumors showed reduced p16INK4a protein and reduced senescence markers, confirming susceptibility to transformation. Superficial GEM-PNST contained regions of nerve-associated plexiform neurofibromas or atypical neurofibromas and grew rapidly on transplantation. Transcriptome analyses showed similarities to corresponding human tumors. Thus, we recapitulated nerve tumor progression in NF1 and provided preclinical platforms for testing therapies at each tumor grade. These results support a tumor progression model in which loss of NF1 in Schwann cells drives plexiform neurofibromas formation, additional loss of Ink4a/Arf contributes to atypical neurofibromas formation, and further changes underlie transformation to MPNST. SIGNIFICANCE: New mouse models recapitulate the stepwise progression of NF1 tumors and will be useful to define effective treatments that halt tumor growth and tumor progression in NF1.

Phase separation in necrotic cells.

Necrotic cells are known to develop characteristic membrane blebs. We measured protein concentration within necrotic blebs and found that it can be reduced by as much as twenty-fold compared to the main cell body (CB). These results raise two questions: 1. Why do proteins vacate the bleb? 2. How can osmotic equilibrium be maintained between the bleb and CB? Our photobleaching and ultracentrifugation experiments indicate extensive protein aggregation. We hypothesize that protein aggregation within the CB shifts the chemical equilibrium and draws proteins out of the bleb; at the same time, aggregation reduces the effective molar concentration of protein in the CB, so that osmotic equilibrium between high-protein CB and low-protein necrotic blebs becomes possible.

RACK1 regulates neural development.

Receptor for activated C kinase 1 (RACK1) is an evolutionarily conserved scaffolding protein within the tryptophan-aspartate (WD) repeat family of proteins. RACK1 can bind multiple signaling molecules concurrently, as well as stabilize and anchor proteins. RACK1 also plays an important role at focal adhesions, where it acts to regulate cell migration. In addition, RACK1 is a ribosomal binding protein and thus, regulates translation. Despite these numerous functions, little is known about how RACK1 regulates nervous system development. Here, we review three studies that examine the role of RACK1 in neural development. In brief, these papers demonstrate that (1) RACK-1, the C. elegans homolog of mammalian RACK1, is required for axon guidance; (2) RACK1 is required for neurite extension of neuronally differentiated rat PC12 cells; and (3) RACK1 is required for axon outgrowth of primary mouse cortical neurons. Thus, it is evident that RACK1 is critical for appropriate neural development in a wide range of species, and future discoveries could reveal whether RACK1 and its signaling partners are potential targets for treatment of neurodevelopmental disorders or a therapeutic approach for axonal regeneration.

RACK1 is necessary for the formation of point contacts and regulates axon growth.

Receptor for activated C kinase 1 (RACK1) is a multifunctional ribosomal scaffolding protein that can interact with multiple signaling molecules concurrently through its seven WD40 repeats. We recently found that RACK1 is localized to mammalian growth cones, prompting an investigation into its role during neural development. Here, we show for the first time that RACK1 localizes to point contacts within mouse cortical growth cones. Point contacts are adhesion sites that link the actin network within growth cones to the extracellular matrix, and are necessary for appropriate axon guidance. Our experiments show that RACK1 is necessary for point contact formation. Brain-derived neurotrophic factor (BDNF) stimulates an increase in point contact density, which was eliminated by RACK1 shRNA or overexpression of a nonphosphorylatable mutant form of RACK1. We also found that axonal growth requires both RACK1 expression and phosphorylation. We have previously shown that the local translation of ß-actin mRNA within growth cones is necessary for appropriate axon guidance and is dependent on RACK1. Thus, we examined the location of members of the local translation complex relative to point contacts. Indeed, both ß-actin mRNA and RACK1 colocalize with point contacts, and this colocalization increases following BDNF stimulation. This implies the novel finding that local translation is regulated at point contacts. Taken together, these data suggest that point contacts are a targeted site of local translation within growth cones, and RACK1 is a critical member of the point contact complex and necessary for appropriate neural development. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1038-1056, 2017.