Formation and regulation of excitatory glutamatergic synapses is essential for shaping neural circuits throughout development. In a Drosophila genetic screen for synaptogenesis mutants, we identified mind the gap (mtg), which encodes a secreted, extracellular N-glycosaminoglycan-binding protein. MTG is expressed neuronally and detected in the synaptic cleft, and is required to form the specialized transsynaptic matrix that links the presynaptic active zone with the post-synaptic glutamate receptor (GluR) domain. Null mtg embryonic mutant synapses exhibit greatly reduced GluR function, and a corresponding loss of localized GluR domains. All known post-synaptic signaling/scaffold proteins functioning upstream of GluR localization are also grossly reduced or mislocalized in mtg mutants, including the dPix-dPak-Dock cascade and the Dlg/PSD-95 scaffold. Ubiquitous or neuronally targeted mtg RNA interference (RNAi) similarly reduce post-synaptic assembly, whereas post-synaptically targeted RNAi has no effect, indicating that presynaptic MTG induces and maintains the post-synaptic pathways driving GluR domain formation. These findings suggest that MTG is secreted from the presynaptic terminal to shape the extracellular synaptic cleft domain, and that the cleft domain functions to mediate transsynaptic signals required for post-synaptic development.[Keywords: Glutamatergic synaptogenesis; post-synaptic density; glutamate receptor; secretion; Drosophila; neuromuscular junction] Supplemental material is available at http://www.genesdev.org.

Purpose: Glioblastomas (GBMs), neoplasms derived from glia and neuroglial progenitor cells, are the most common and lethal malignant primary brain tumors diagnosed in adults, with a median survival of 14 months. GBM tumorigenicity is often driven by genetic aberrations in receptor tyrosine kinases, such as amplification and mutation of EGFR. Experimental Design: Using a Drosophila glioma model and human patient–derived GBM stem cells and xenograft models, we genetically and pharmacologically tested whether the YAP and TAZ transcription coactivators, effectors of the Hippo pathway that promote gene expression via TEA domain (TEAD) cofactors, are key drivers of GBM tumorigenicity downstream of oncogenic EGFR signaling. Results: YAP and TAZ are highly expressed in EGFR-amplified/mutant human GBMs, and their knockdown in EGFR-amplified/mutant GBM cells inhibited proliferation and elicited apoptosis. Our results indicate that YAP/TAZ-TEAD directly regulates transcription of SOX2, C-MYC, and EGFR itself to create a feedforward loop to drive survival and proliferation of human GBM cells. Moreover, the benzoporphyrin derivative verteporfin, a disruptor of YAP/TAZ-TEAD–mediated transcription, preferentially induced apoptosis of cultured patient-derived EGFR-amplified/mutant GBM cells, suppressed expression of YAP/TAZ transcriptional targets, including EGFR, and conferred significant survival benefit in an orthotopic xenograft GBM model. Our efforts led us to design and initiate a phase 0 clinical trial of Visudyne, an FDA-approved liposomal formulation of verteporfin, where we used intraoperative fluorescence to observe verteporfin uptake into tumor cells in GBM tumors in human patients. Conclusions: Together, our data suggest that verteporfin is a promising therapeutic agent for EGFR-amplified and -mutant GBM.

Glioblastoma (GBM) and lower grade gliomas (LGG) are the most common primary malignant brain tumors and are resistant to current therapies. Genomic analyses reveal that signature genetic lesions in GBM and LGG include copy gain and amplification of chromosome 7, amplification, mutation, and overexpression of receptor tyrosine kinases (RTK) such as EGFR, and activating mutations in components of the PI3K pathway. In Drosophila melanogaster, constitutive co-activation of RTK and PI3K signaling in glial progenitor cells recapitulates key features of human gliomas. Here we use this Drosophila glioma model to identify death-associated protein kinase (Drak), a cytoplasmic serine/threonine kinase orthologous to the human kinase STK17A, as a downstream effector of EGFR and PI3K signaling pathways. Drak was necessary for glial neoplasia, but not for normal glial proliferation and development, and Drak cooperated with EGFR to promote glial cell transformation. Drak phosphorylated Sqh, the Drosophila ortholog of nonmuscle myosin regulatory light chain (MRLC), which was necessary for transformation. Moreover, Anillin, which is a binding partner of phosphorylated Sqh, was upregulated in a Drak-dependent manner in mitotic cells and colocalized with phosphorylated Sqh in neoplastic cells undergoing mitosis and cytokinesis, consistent with their known roles in nonmuscle myosin-dependent cytokinesis. These functional relationships were conserved in human GBM. Our results indicate that Drak/STK17A, its substrate Sqh/MRLC, and the effector Anillin/ANLN regulate mitosis and cytokinesis in gliomas. This pathway may provide a new therapeutic target for gliomas. Significance: These findings reveal new insights into differential regulation of cell proliferation in malignant brain tumors, which will have a broader impact on research regarding mechanisms of oncogene cooperation and dependencies in cancer. See related commentary by Lathia, p. 1036

Object Tissue engineering appears to be a promising strategy for articular cartilage regeneration as a treatment for facet joint arthritis. Prior to the commencement of tissue engineering approaches, design criteria must be established to determine the required functional properties of the replacement tissue. As characterization of the functional properties of facet joint cartilage has not been performed previously, the objective of this study was to determine the biomechanical, biochemical, and histological properties of facet joint cartilage. Methods The in vitro testing was conducted using 4 lumbar spinal segments obtained from skeletally mature canines. In each specimen, articular cartilage was obtained from the superior surface of the L3–4 and L4–5 facet joints. Creep indentation was used to determine the compressive biomechanical properties, while uniaxial tensile testing yielded the Young modulus and ultimate tensile strength of the tissue. Additionally, biochemical assessments included determinations of cellularity, glycosaminoglycan (GAG) content, and collagen content, as well as enzymelinked immunosorbent assays for collagen I and II production. Finally, histological characterization included H & E staining, as well as staining for collagen and GAG distributions. Results The means ± standard deviation values were determined. There were no differences between the 2 spinal levels for any of the assessed properties. Averaged over both levels, the thickness was 0.49 ± 0.10 mm and the hydration was 74.7 ± 1.7%. Additionally, the cells/wet weight (WW) ratio was 6.26 ± 2.66 × 104 cells/mg and the cells/dry weight (DW) ratio was 2.51 ± 1.21 × 105 cells/mg. The GAG/WW was 0.038 ± 0.013 and the GAG/ DW was 0.149 ± 0.049 mg/mg, while the collagen/WW was 0.168 ± 0.026 and collagen/DW was 0.681 ± 0.154 mg/ mg. Finally, the aggregate modulus was 554 ± 133 kPa, the Young modulus was 10.08 ± 8.07 MPa, and the ultimate tensile strength was 4.44 ± 2.40 MPa. Conclusions To the best of the authors' knowledge, this study is the first to provide a functional characterization of facet joint articular cartilage, thus providing design criteria for future tissue engineering studies.