Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4 KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF FARP2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/FARP2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs, highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.
Background ATRX inactivation occurs with IDH1 R132H and p53 mutations in over 80% of Grade II/III astrocytomas. It is believed that ATRX loss contributes to oncogenesis by dysregulating epigenetic and telomere mechanisms but effects on anti-glioma immunity have not been explored. This paper examines how ATRX loss contributes to the malignant and immunosuppressive phenotypes of IDH1 R132H/p53mut glioma cells and xenografts. Methods Isogenic astrocytoma cells (+/-IDH1 R132H/+/-ATRXloss) were established in p53mut astrocytoma cell lines using lentivirus encoding doxycycline inducible IDH1 R132H, ATRX shRNA or Lenti-CRISPR/Cas9 ATRX. Effects of IDH1 R132H+/- ATRXloss on cell migration, growth, DNA repair and tumorigenicity were evaluated by clonal growth, transwell and scratch assays, MTT, immunofluorence and immunoblotting assays and xenograft growth. Effects on the expression and function of modulators of the immune microenvironment were quantified by qRT-PCR, immunoblot, T-cell function, macrophage polarization and flow cytometry assays. Pharmacologic inhibitors were used to examine epigenetic drivers of the immunosuppressive transcriptome of IDH1 R132H/p53mut/ATRXloss cells. Results Adding ATRX loss to the IDH1 R132H/p53mut background promoted astrocytoma cell aggressiveness, induced expression of BET proteins BRD3/4 and an immune suppressive transcriptome consisting of up-regulated immune checkpoints (e.g. PD-L1, PD-L2) and altered cytokine/chemokine profiles (e.g. IL33, CXCL8, CSF2, IL6, CXCL9). ATRX loss enhanced the capacity of IDH1 R132H/p53mut cells to induce T-cell apoptosis, tumorigenic/anti-inflammatory macrophage polarization and Treg infiltration. The transcriptional and biological immune suppressive responses to ATRX loss were enhanced by temozolomide and radiation and abrogated by pharmacologic BET inhibition. Conclusions ATRX loss activates a BRD-dependent immune suppressive transcriptome and immune escape mechanism in IDH1 R132H/p53mut astrocytoma cells.
Diverse neuronal populations with distinct cellular morphologies coordinate the complex function of the nervous system. Establishment of distinct neuronal morphologies critically depends on signaling pathways that control axonal and dendritic development. The Sema3A-Nrp1/PlxnA4 signaling pathway promotes cortical neuron basal dendrite arborization but also repels axons. However, the downstream signaling components underlying these disparate functions of Sema3A signaling are unclear. Using the novel PlxnA4KRK-AAA knock-in male and female mice, generated by CRISPR/cas9, we show here that the KRK motif in the PlxnA4 cytoplasmic domain is required for Sema3A-mediated cortical neuron dendritic elaboration but is dispensable for inhibitory axon guidance. The RhoGEF Farp2, which binds to the KRK motif, shows identical functional specificity as the KRK motif in the PlxnA4 receptor. We find that Sema3A activates the small GTPase Rac1, and that Rac1 activity is required for dendrite elaboration but not axon growth cone collapse. This work identifies a novel Sema3A-Nrp1/PlxnA4/Farp2/Rac1 signaling pathway that specifically controls dendritic morphogenesis but is dispensable for repulsive guidance events. Overall, our results demonstrate that the divergent signaling output from multifunctional receptor complexes critically depends on distinct signaling motifs highlighting the modular nature of guidance cue receptors and its potential to regulate diverse cellular responses.Significance StatementThe proper formation of axonal and dendritic morphologies is crucial for the precise wiring of the nervous system that ultimately leads to the generation of complex functions in an organism. The Semaphorin3A-Neuropilin1/PlexinA4 signaling pathway has been shown to have multiple key roles in neurodevelopment, from axon repulsion to dendrite elaboration. This study demonstrates that 3 specific amino acids, the KRK motif within the PlexinA4 receptor cytoplasmic domain, are required to coordinate the downstream signaling molecules to promote Sema3A-mediated cortical neuron dendritic elaboration, but not inhibitory axon guidance. Our results unravel a novel Semaphorin3A-PlexinA4 downstream signaling pathway and shed light on how the disparate functions of axon guidance and dendritic morphogenesis are accomplished by the same extracellular ligand in vivo.
OBJECTIVEMetric tracking of grant funding over time for academic neurosurgeons sorted by gender informs the current climate of career development internationally for women in neurosurgery.METHODSMultivariate linear trend analysis of grant funding awarded to neurosurgeons in the NIH and World Research Portfolio Online Reporting Tools Expenditures and Results (RePORTER) was performed. Traveling fellowships for international neurosurgery residents sponsored by the AANS and Congress of Neurological Surgeons (CNS) were also analyzed.RESULTSWithin the US, funding awarded to female neurosurgeons has remained static from 2009 to 2019 after adjusting for inflation and overall trends in NIH funding (β = −$0.3 million per year, p = 0.16). Internationally, female neurosurgeons represented 21.7% (n = 5) of project leads for World RePORTER grants. Traveling fellowships are also an important building block for young international female neurosurgeons, of which 7.4% (n = 2) of AANS international traveling fellowships and 19.4% (n = 7) of AANS/CNS pediatrics international traveling fellowships are women.CONCLUSIONSOver the past decade, funding has increased in neurosurgery without a concordant increase in funding awarded to women. Recognition of this trend is essential to focus efforts on research and career development opportunities for women in neurosurgery. Worldwide, female neurosurgeons head one-fifth of the funded project leads and constitute a minority of international traveling fellowships awarded by organized neurosurgery.
OBJECTIVE At present, females constitute less than 10% of neurosurgeons in the US, despite representing approximately half of all medical students. Multiple barriers have been described for females entering the neurosurgical field, particularly academic neurosurgery. Understanding the environment that female neurosurgeons face and any potential barriers preventing career advancement is needed to recruit, promote, and retain females in neurosurgery. METHODS The gender composition of editorial boards for 5 high-impact neurosurgery journals was analyzed from 2000 to 2020. The names of editorial board members were obtained directly from the journal administration, physical copies of the published journal, or publicly available data through each journal’s website. The gender, degrees, academic titles, H-index, and country were determined for each individual and statistical tests were performed to identify significant differences. RESULTS Of the 466 identified individuals that served on at least one editorial board between 2000 and 2020, there were 36 females (7.7%) and 430 males (92.3%). There were no significant differences between males and females serving on multiple editorial boards. Most females possessed an additional graduate degree (58.3%), while only one-third of males (33.5%) obtained such a degree (p = 0.002). In addition, males had significantly higher average H-indices than females (p = 0.002). These trends were also observed when analyzing only US-based editorial board members. Although females were more likely overall to be identified as associate professors, males were more likely to be appointed as full professors (p = 0.001); this trend did not remain true in the US-based cohort. When analyzing the editorial boards for individual journals, all 5 journals experienced an increase of female representation since 2000 or since their inception after 2000. The highest proportion of females for a single journal was 27.3% in 2020. All other journals ranged from 11.0% to 13.5% in 2020. CONCLUSIONS When entering the field of neurosurgery, females continue to face significant social and academic barriers. While the proportion of females on editorial boards for neurosurgery journals in 2020 is consistent with the proportion of practicing female neurosurgeons, there is a statistically significantly higher likelihood that females possess additional graduate degrees and lower H-indices compared to their male counterparts. The authors encourage neurosurgical journals to continue expanding female representation on editorial boards.
Four different molecular subgroups are recognized in medulloblastoma (MB). Among these subgroups, Group 3 and 4 tumors are the most aggressive malignancies with few effective therapies. New therapies are likely to be most effective if they target molecular alterations that mediate the formation and growth of these malignancies. We searched the public cancer microarray database Oncomine and found that the expression of OTX2, c-Myc, polycomb repressor complex 2 (PRC2) subunits EZH2 and SuZ12 are significantly higher in Groups 3 and 4 as compared to the SHH subgroups. Moreover, Immunohistochemical staining of a medulloblastoma tissue array showed that H3k27me3 expression is higher in Group 3 and 4 compared with the SHH groups. These findings suggest that these distinct molecular alterations could represent promising therapeutic targets. In this study, we identify miRNAs with the potential to serve as therapeutic agents for Groups 3 and 4 by targeting oncogene pathway OTX2 and c-Myc as well as targeting PRC2 and modulating H3k27me3-targeted tumor suppressors. Using TargetScan, Pictar and miRanda prediction algorithms, miR-148a-3p is predicted to target OTX2. We show that enforced miR-148a-3p potently inhibits protein expression of OTX2 and c-Myc in medulloblastoma cells. Luciferase reporter analysis shows that miR-148a-3p expression downregulates OTX2 luciferase activities, and overexpressing OTX2 cDNA that lack the respective 3’-UTR region recognized by miR-148a-3p significantly rescues OTX2 downregulation by miR-148a-3p. Furthermore, we show for the first time that enforced miR-148a-3p expression can down-regulate the expression of the PRC2 methylase EZH2 and the PRC2 regulatory subunit SUZ12 in multiple MB cell lines. Conversely, silencing endogenous miR-148a-3p expression up-regulates the expression of EZH2 and SUZ12. Furthermore, enforced OTX2 expression attenuates the expression of miR-148a-3p-downregulated PRC2 subunit EZh2 and Suz12. Consistent with the effects of miR-148a-3p on EZH2 and SUZ12, miR-148a-3p was found to modulate H3k27me3 levels and restore the expression of H3k27me3-targeted tumor suppressors. We also show that enforced miR-148a-3p increases the sensitivity of Group 3 and 4 medulloblastoma cells to radiation and chemotherapy. Together, our findings show that miR-148a-3p has the potential to modulate Group 3 and 4 medulluloblastoma malignancy and chemo/radiation sensitivity by (1) reversing H3k27me3 modifications and thereby potentially restoring tumor suppressor expression; and (2) down-regulating OTX2 and leading to the inhibition of c-Myc-dependent oncogenes. Citation Format: Qingfu Xu, Kimberly Wang, Sama Ahsan, Fausto Rodriguez, Charles Eberhart, Yugang Jiang, John Laterra, Yunqing Li. miR-148a-3p regulation of OTX2 and PRC2 in group 3 and 4 medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1471. doi:10.1158/1538-7445.AM2017-1471
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