Gcap14 is a microtubule plus-end-tracking protein coordinating microtubule-actin crosstalk during neurodevelopment.

Regulation of microtubule dynamics is required to properly control various steps of neurodevelopment. In this study, we identified granule cell antiserum-positive 14 (Gcap14) as a microtubule plus-end-tracking protein and as a regulator of microtubule dynamics during neurodevelopment. Gcap14 knockout mice exhibited impaired cortical lamination. Gcap14 deficiency resulted in defective neuronal migration. Moreover, nuclear distribution element nudE-like 1 (Ndel1), an interacting partner of Gcap14, effectively corrected the downregulation of microtubule dynamics and the defects in neuronal migration caused by Gcap14 deficiency. Finally, we found that the Gcap14-Ndel1 complex participates in the functional link between microtubule and actin filament, thereby regulating their crosstalks in the growth cones of cortical neurons. Taken together, we propose that the Gcap14-Ndel1 complex is fundamental for cytoskeletal remodeling during neurodevelopmental processes such as neuronal processes elongation and neuronal migration.

Schizophrenia-associated Mitotic Arrest Deficient-1 (MAD1) regulates the polarity of migrating neurons in the developing neocortex.

Although large-scale genome-wide association studies (GWAS) have identified an association between MAD1L1 (Mitotic Arrest Deficient-1 Like 1) and the pathology of schizophrenia, the molecular mechanisms underlying this association remain unclear. In the present study, we aimed to address these mechanisms by examining the role of MAD1 (the gene product of MAD1L1) in key neurodevelopmental processes in mice and human organoids. Our findings indicated that MAD1 is highly expressed during active cortical development and that MAD1 deficiency leads to impairments in neuronal migration and neurite outgrowth. We also observed that MAD1 is localized to the Golgi apparatus and regulates vesicular trafficking from the Golgi apparatus to the plasma membrane, which is required for the growth and polarity of migrating neurons. In this process, MAD1 physically interacts and collaborates with the kinesin-like protein KIFC3 (kinesin family member C3) to regulate the morphology of the Golgi apparatus and neuronal polarity, thereby ensuring proper neuronal migration and differentiation. Consequently, our findings indicate that MAD1 is an essential regulator of neuronal development and that alterations in MAD1 may underlie schizophrenia pathobiology.

Loss of the neural-specific BAF subunit ACTL6B relieves repression of early response genes and causes recessive autism.

Synaptic activity in neurons leads to the rapid activation of genes involved in mammalian behavior. ATP-dependent chromatin remodelers such as the BAF complex contribute to these responses and are generally thought to activate transcription. However, the mechanisms keeping such "early activation" genes silent have been a mystery. In the course of investigating Mendelian recessive autism, we identified six families with segregating loss-of-function mutations in the neuronal BAF (nBAF) subunit ACTL6B (originally named BAF53b). Accordingly, ACTL6B was the most significantly mutated gene in the Simons Recessive Autism Cohort. At least 14 subunits of the nBAF complex are mutated in autism, collectively making it a major contributor to autism spectrum disorder (ASD). Patient mutations destabilized ACTL6B protein in neurons and rerouted dendrites to the wrong glomerulus in the fly olfactory system. Humans and mice lacking ACTL6B showed corpus callosum hypoplasia, indicating a conserved role for ACTL6B in facilitating neural connectivity. Actl6b knockout mice on two genetic backgrounds exhibited ASD-related behaviors, including social and memory impairments, repetitive behaviors, and hyperactivity. Surprisingly, mutation of Actl6b relieved repression of early response genes including AP1 transcription factors (Fos, Fosl2, Fosb, and Junb), increased chromatin accessibility at AP1 binding sites, and transcriptional changes in late response genes associated with early response transcription factor activity. ACTL6B loss is thus an important cause of recessive ASD, with impaired neuron-specific chromatin repression indicated as a potential mechanism.

Nf1 limits epicardial derivative expansion by regulating epithelial to mesenchymal transition and proliferation.

The epicardium is the primary source of coronary vascular smooth muscle cells (cVSMCs) and fibroblasts that reside in the compact myocardium. To form these epicardial-derived cells (EPDCs), the epicardium undergoes the process of epithelial to mesenchymal transition (EMT). Although several signaling pathways have been identified that disrupt EMT, no pathway has been reported that restricts this developmental process. Here, we identify neurofibromin 1 (Nf1) as a key mediator of epicardial EMT. To determine the function of Nf1 during epicardial EMT and the formation of epicardial derivatives, cardiac fibroblasts and cVSMCs, we generated mice with a tissue-specific deletion of Nf1 in the epicardium. We found that mutant epicardial cells transitioned more readily to mesenchymal cells in vitro and in vivo. The mesothelial epicardium lost epithelial gene expression and became more invasive. Using lineage tracing of EPDCs, we found that the process of EMT occurred earlier in Nf1 mutant hearts, with an increase in epicardial cells entering the compact myocardium. Moreover, loss of Nf1 caused increased EPDC proliferation and resulted in more cardiac fibroblasts and cVSMCs. Finally, we were able to partially reverse the excessive EMT caused by loss of Nf1 by disrupting Pdgfrα expression in the epicardium. Conversely, Nf1 activation was able to inhibit PDGF-induced epicardial EMT. Our results demonstrate a regulatory role for Nf1 during epicardial EMT and provide insights into the susceptibility of patients with disrupted NF1 signaling to cardiovascular disease.

The bHLH transcription factor Tcf21 is required for lineage-specific EMT of cardiac fibroblast progenitors.

The basic helix-loop-helix (bHLH) family of transcription factors orchestrates cell-fate specification, commitment and differentiation in multiple cell lineages during development. Here, we describe the role of a bHLH transcription factor, Tcf21 (epicardin/Pod1/capsulin), in specification of the cardiac fibroblast lineage. In the developing heart, the epicardium constitutes the primary source of progenitor cells that form two cell lineages: coronary vascular smooth muscle cells (cVSMCs) and cardiac fibroblasts. Currently, there is a debate regarding whether the specification of these lineages occurs early in the formation of the epicardium or later after the cells have entered the myocardium. Lineage tracing using a tamoxifen-inducible Cre expressed from the Tcf21 locus demonstrated that the majority of Tcf21-expressing epicardial cells are committed to the cardiac fibroblast lineage prior to initiation of epicardial epithelial-to-mesenchymal transition (EMT). Furthermore, Tcf21 null hearts fail to form cardiac fibroblasts, and lineage tracing of the null cells showed their inability to undergo EMT. This is the first report of a transcription factor essential for the development of cardiac fibroblasts. We demonstrate a unique role for Tcf21 in multipotent epicardial progenitors, prior to the process of EMT that is essential for cardiac fibroblast development.

Impairments of cerebellar structure and function in a zebrafish KO of neuropsychiatric risk gene znf536.

Genetic variants in ZNF536 contribute to the risk for neuropsychiatric disorders such as schizophrenia, autism, and others. The role of this putative transcriptional repressor in brain development and function is, however, largely unknown. We generated znf536 knockout (KO) zebrafish and studied their behavior, brain anatomy, and brain function. Larval KO zebrafish showed a reduced ability to compete for food, resulting in decreased total body length and size. This phenotype can be rescued by segregating the homozygous KO larvae from their wild-type and heterozygous siblings, enabling studies of adult homozygous KO animals. In adult KO zebrafish, we observed significant reductions in anxiety-like behavior and social interaction. These znf536 KO zebrafish have decreased cerebellar volume, corresponding to decreased populations of specific neuronal cells, especially in the valvular cerebelli (Va). Finally, using a Tg[mbp:mgfp] line, we identified a previously undetected myelin structure located bilaterally within the Va, which also displayed a reduction in volume and disorganization in KO zebrafish. These findings indicate an important role for ZNF536 in brain development and implicate the cerebellum in the pathophysiology of neuropsychiatric disorders.

Broadening the scope of multigene panel analysis for adult epilepsy patients.

OBJECTIVE: Epilepsy is a suitable target for gene panel sequencing because a considerable portion of epilepsy is now explained by genetic components, especially in syndromic cases. However, previous gene panel studies on epilepsy have mostly focused on pediatric patients. METHODS: We enrolled adult epilepsy patients meeting any of the following criteria: family history of epilepsy, seizure onset age ≤ 19 years, neuronal migration disorder, and seizure freedom not achieved by dual anti-seizure medications. We sequenced the exonic regions of 211 epilepsy genes in these patients. To confirm the pathogenicity of a novel MTOR truncating variant, we electroporated vectors with different MTOR variants into developing mouse brains. RESULTS: A total of 92 probands and 4 affected relatives were tested, and the proportion of intellectual disability (ID) and/or developmental disability (DD) was 21.7%. As a result, twelve probands (13.0%) had pathogenic or likely pathogenic variants in the following genes or regions: DEPDC5, 15q12-q13 duplication (n = 2), SLC6A1, SYNGAP1, EEF1A2, LGI1, MTOR, KCNQ2, MEF2C, and TSC1 (n = 1). We confirmed the functional impact of a novel truncating mutation in the MTOR gene (c.7570C > T, p.Gln2524Ter) that disrupted neuronal migration in a mouse model. The diagnostic yield was higher in patients with ID/DD or childhood-onset seizures. We also identified additional candidate variants in 20 patients that could be reassessed by further studies. SIGNIFICANCE: Our findings underscore the clinical utility of gene panel sequencing in adult epilepsy patients suspected of having genetic etiology, especially those with ID/DD or early-onset seizures. Gene panel sequencing could not only lead to genetic diagnosis in a substantial portion of adult epilepsy patients but also inform more precise therapeutic decisions based on their genetic background. PLAIN LANGUAGE SUMMARY: This study demonstrated the effectiveness of gene panel sequencing in adults with epilepsy, revealing pathogenic or likely pathogenic variants in 13.0% of patients. Higher diagnostic yields were observed in those with neurodevelopmental disorders or childhood-onset seizures. Additionally, we have shown that expanding genetic studies into adult patients would uncover new types of pathogenic variants for epilepsy, contributing to the advancement of precision medicine for individuals with epilepsy. In conclusion, our results highlight the practical value of employing gene panel sequencing in adult epilepsy patients, particularly when genetic etiology is clinically suspected.

Targeting phosphorylation circuits on CREB and CRTCs as the strategy to prevent acquired skin hyperpigmentation.

Background: The cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivators (CRTCs) cooperate in the transcriptional activation of microphthalmia-associated transcription factor subtype M (MITF-M) that is a master regulator in the biogenesis, pigmentation and transfer of melanosomes at epidermal melanocytes. Here, we propose the targeting of phosphorylation circuits on CREB and CRTCs in the expression of MITF-M as the rationale to prevent skin hyperpigmentation by elucidating the inhibitory activity and mechanism of yakuchinone A (Yaku A) on facultative melanogenesis. Methods: We employed human epidermal melanocyte cell, mouse skin, and mouse melanoma cell, and applied Western blotting, reverse transcription-polymerase chain reaction, immunoprecipitation and confocal microscopy to conduct this study. Results: This study suggested that α-melanocyte stimulating hormone (α-MSH)-induced melanogenic programs could switch on the axis of protein kinase A-salt inducible kinases (PKA-SIKs) rather than that of PKA-AMP activated protein kinase (PKA-AMPK) during the dephosphorylation of CRTCs in the expression of MITF-M. SIK inhibitors rather than AMPK inhibitors stimulated melanin production in melanocyte cultures in the absence of extracellular melanogenic stimuli, wherein SIK inhibitors increased the dephosphorylation of CRTCs but bypassed the phosphorylation of CREB for the expression of MITF-M. Treatment with Yaku A prevented ultraviolet B (UV-B)-irradiated skin hyperpigmentation in mice and inhibited melanin production in α-MSH- or SIK inhibitor-activated melanocyte cultures. Mechanistically, Yaku A suppressed the expression of MITF-M via dually targeting the i) cAMP-dependent dissociation of PKA holoenzyme at the upstream from PKA-catalyzed phosphorylation of CREB coupled with PKA-SIKs axis-mediated dephosphorylation of CRTCs in α-MSH-induced melanogenic programs, and ii) nuclear import of CRTCs after SIK inhibitor-induced dephosphorylation of CRTCs. Conclusions: Taken together, the targeting phosphorylation circuits on CREB and CRTCs in the expression of MITF-M could be a suitable strategy to prevent pigmentary disorders in the skin.

Hemimegalencephaly, a paradigm for somatic postzygotic neurodevelopmental disorders.

PURPOSE OF REVIEW: Combining human genomics and molecular biology, recent studies have made pivotal progress toward understanding the cause of hemimegalencephaly (HME) and other cerebral megalencephaly syndromes. The present article highlights recent advances of the genetic cause of these conditions, and considers the role of somatic postzygotic genetic lesions in brain maldevelopment. RECENT FINDINGS: Studies over the past 12 months have identified de-novo somatic mutations as one possible cause in HME. The gene mutations involve components of the phosphatidylinositol 3-kinase (PI3K)-AKT (also known as protein kinase B)-mammalian target of rapamycin (mTOR) pathway and include PIK3CA, PIK3R2, AKT3, and MTOR. These mutations were identified by comparing genomic data obtained from surgically resected brain tissue with nondiseased tissue, and by single-neuron sequencing in combination with molecular biology techniques. The association between the somatic mutations and downstream activation of the PI3K-mTOR pathway suggests that HME is a neurodevelopmental disease caused by gain-of-function activation of the PI3K-AKT-mTOR pathway. SUMMARY: The studies reviewed suggest that somatic mutations of the PI3K-AKT-mTOR pathway limited to the brain may represent one cause of HME. Dysregulation of this pathway has possible therapeutic potential in the identification of HME. Somatic mutations may be an important yet underappreciated disease mechanism in developmental neurological diseases.

Epicardial-derived cell epithelial-to-mesenchymal transition and fate specification require PDGF receptor signaling.

RATIONALE: In early heart development, platelet-derived growth factor (PDGF) receptor expression in the heart ventricles is restricted to the epicardium. Previously, we showed that PDGFRß is required for coronary vascular smooth muscle cell (cVSMC) development, but a role for PDGFRα has not been identified. Therefore, we investigated the combined and independent roles of these receptors in epicardial development. OBJECTIVE: To understand the contribution of PDGF receptors in epicardial development and epicardial-derived cell fate determination. METHODS AND RESULTS: By generating mice with epicardial-specific deletion of the PDGF receptors, we found that epicardial epithelial-to-mesenchymal transition (EMT) was defective. Sox9, an SRY-related transcription factor, was reduced in PDGF receptor-deficient epicardial cells, and overexpression of Sox9 restored epicardial migration, actin reorganization, and EMT gene expression profiles. The failure of epicardial EMT resulted in hearts that lacked epicardial-derived cardiac fibroblasts and cVSMC. Loss of PDGFRα resulted in a specific disruption of cardiac fibroblast development, whereas cVSMC development was unperturbed. CONCLUSIONS: Signaling through both PDGF receptors is necessary for epicardial EMT and formation of epicardial-mesenchymal derivatives. PDGF receptors also have independent functions in the development of specific epicardial-derived cell fates.

Targeting Wnt/ß-catenin-mediated upregulation of oncogenic NLGN3 suppresses cancer stem cells in glioblastoma.

Glioblastoma (GBM) is the most malignant tumor in brain and is highly resistant to therapy. Clinical evidence suggests increased number of cancer stem cells (CSCs) may contribute to the failure of conventional therapies, but the mechanisms associated with acquisition of CSC properties in GBM are not fully understood. We found that DAB2IP suppresses CSC properties by targeting the synaptic proteins neuroligin 3 (NLGN3) in GBM. Furthermore, we showed that GBM-derived NLGN3 has an oncogenic function by inducing CSC properties within GBM. Moreover, elevated NLGN3 transcription mediated by Wnt/ß-catenin signaling pathway resulted in increased secretion of NLGN3 into the surrounding tumor microenvironment. Both condition media containing NLGN3 and recombinant NLGN3 transformed neighboring cells to CSCs, suggesting NLGN3 as a critical component inducing CSC properties. Furthermore, targeting NLGN3-bearing CSCs using upstream Wnt/ß-catenin inhibitors synergistically enhances the efficacy of conventional treatment. Hence, we unveiled the series of regulatory mechanisms for acquisition of CSC properties in GBM progression by Wnt/ß-catenin-mediated NLGN3. These results may provide a new targeting strategy to improve the therapeutic efficacy of GBM treatments.

Reversibility and developmental neuropathology of linear nevus sebaceous syndrome caused by dysregulation of the RAS pathway.

Linear nevus sebaceous syndrome (LNSS) is a neurocutaneous disorder caused by somatic gain-of-function mutations in KRAS or HRAS. LNSS brains have neurodevelopmental defects, including cerebral defects and epilepsy; however, its pathological mechanism and potentials for treatment are largely unclear. We show that introduction of KRASG12V in the developing mouse cortex results in subcortical nodular heterotopia and enhanced excitability, recapitulating major pathological manifestations of LNSS. Moreover, we show that decreased firing frequency of inhibitory neurons without KRASG12V expression leads to disrupted excitation and inhibition balance. Transcriptional profiling after destabilization domain-mediated clearance of KRASG12V in human neural progenitors and differentiating neurons identifies reversible functional networks underlying LNSS. Neurons expressing KRASG12V show molecular changes associated with delayed neuronal maturation, most of which are restored by KRASG12V clearance. These findings provide insights into the molecular networks underlying the reversibility of some of the neuropathologies observed in LNSS caused by dysregulation of the RAS pathway.

Detecting Low-Variant Allele Frequency Mosaic Pathogenic Variants of NF1, TSC2, and AKT3 Genes from Blood in Patients with Neurodevelopmental Disorders.

Growing evidence indicates that early and late postzygotic mosaicism can cause neurodevelopmental disorders (NDDs), but detection of low variant allele frequency (VAF) mosaic variants from blood remains a challenge. Data of 2162 patients with NDDs who underwent conventional genetic tests were reviewed and a deep sequencing was performed using a specifically designed mosaic next-generation sequencing (NGS) panel in the patients with negative genetic test results. Forty-four patents with neurocutaneous syndrome, malformation of cortical development, or nonlesional epileptic encephalopathies were included. In total, mosaic variants were detected from blood in 1.2% (25/2162) of the patients. Using conventional NGS panels, 22 mosaic variants (VAF, 8.8% to 29.8%) were identified in 18 different genes. Using a specifically designed mosaicism NGS panel, three mosaic variants of the NF1, TSC2, and AKT3 genes were identified (VAF, 2.0% to 11.2%). Mosaic variants were found frequently in the patients who had neurocutaneous syndrome (2/7, 28.6%), whereas only one or no mosaic variant was detected for patients who had malformations of cortical development (1/20, 5%) or nonlesional epileptic encephalopathies (0%, 0/17). In summary, mosaic variants that contribute to the spectrum of NDDs can be detected from blood via conventional NGS and specifically designed mosaicism NGS panels, and detection of mosaic variants using blood will increase diagnostic yield.

Stanniocalcin 2 drives malignant transformation of human glioblastoma cells by targeting SNAI2 and Matrix Metalloproteinases.

Glioblastoma multiforme (GBM) is the most malignant brain tumor and is refractory to conventional therapies. Although previous studies have proposed that the interaction between gene mutations and the external environment leads to the occurrence of GBM, the pathogenesis of GBM is still unclear and much remains to be studied. Herein, we show an association between human glycoprotein stanniocalcin-2 (STC2) and aggressive GBM progression, and demonstrate the underlying mechanism. Elevated STC2 expression and secretion greatly increase GBM cell growth and invasive phenotypes. Mechanistically, both, conditioned media (CM) containing STC2 and recombinant STC2, can induce the transformation of GBM cells into more malignant phenotypes by upregulating the expression of the epithelial-mesenchymal transition transcription factor, snail family transcription repressor 2 (SNAI2) as well as matrix metalloproteinases (MMPs). Moreover, we further demonstrate that the oncogenic function of STC2 in GBM is mediated through the MAPK signaling pathway. Collectively, these results identify the mechanism of STC2 targeting SNAI2 and MMPs through the MAPK pathway in GBM, and provide insights into a potential therapeutic strategy for GBM.

The Korean undiagnosed diseases program phase I: expansion of the nationwide network and the development of long-term infrastructure.

BACKGROUND: Phase I of the Korean Undiagnosed Diseases Program (KUDP), performed for 3 years, has been completed. The Phase I program aimed to solve the problem of undiagnosed patients throughout the country and develop infrastructure, including a data management system and functional core laboratory, for long-term translational research. Herein, we share the clinical experiences of the Phase I program and introduce the activities of the functional core laboratory and data management system. RESULTS: During the program (2018-2020), 458 patients were enrolled and classified into 3 groups according to the following criteria: (I) those with a specific clinical assessment which can be verified by direct testing (32 patients); (II) those with a disease group with genetic and phenotypic heterogeneity (353 patients); and (III) those with atypical presentations or diseases unknown to date (73 patients). All patients underwent individualized diagnostic processes based on the decision of an expert consortium. Confirmative diagnoses were obtained for 242 patients (52.8%). The diagnostic yield was different for each group: 81.3% for Group I, 53.3% for Group II, and 38.4% for Group III. Diagnoses were made by next-generation sequencing for 204 patients (84.3%) and other genetic testing for 35 patients (14.5%). Three patients (1.2%) were diagnosed with nongenetic disorders. The KUDP functional core laboratory, with a group of experts, organized a streamlined research pipeline covering various resources, including animal models, stem cells, structural modeling and metabolic and biochemical approaches. Regular data review was performed to screen for candidate genes among undiagnosed patients, and six different genes were identified for functional research. We also developed a web-based database system that supports clinical cohort management and provides a matchmaker exchange protocol based on a matchbox, likely to reinforce the nationwide clinical network and further international collaboration. CONCLUSIONS: The KUDP evaluated the unmet needs of undiagnosed patients and established infrastructure for a data-sharing system and future functional research. The advancement of the KUDP may lead to sustainable bench-to-bedside research in Korea and contribute to ongoing international collaboration.

Efficient inducible Cre-mediated recombination in Tcf21 cell lineages in the heart and kidney.

Tcf21 is a Class II bHLH family member with essential roles in the formation of the lungs, kidneys, gonads, spleen, and heart. Here, we report the utility of a mouse line with targeted insertion of a tamoxifen-inducible Cre recombinase, MerCreMer at the Tcf21 locus. This mouse line will permit the inducible expression of Cre recombinase in Tcf21-expressing cells. Using ROSA26 reporter mice, we show that Cre recombinase is specifically and robustly activated in multiple Tcf21-expressing tissues during embryonic and postnatal development. The expression profile in the kidney is particularly dynamic with the ability to cause recombination in mesangial cells at one time of induction and podocytes at another time. These features make the Tcf21-driven inducible Cre line (Tcf21(iCre) ) a valuable genetic tool for spatiotemporal gene function analysis and lineage tracing of cells in the heart, kidney, cranial muscle, and gonads.

Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast.

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) 'biological process' terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including 'cell cycle' (cdc2, rik1, pas1, and leo1), 'signaling' (sck2, oga1, and cki3), and 'vesicle-mediated transport' (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the 'signaling' GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Wnt/ß-catenin signaling pathway induces autophagy-mediated temozolomide-resistance in human glioblastoma.

Temozolomide (TMZ) is widely used for treating glioblastoma multiforme (GBM), however, the treatment of such brain tumors remains a challenge due to the development of resistance. Increasing studies have found that TMZ treatment could induce autophagy that may link to therapeutic resistance in GBM, but, the precise mechanisms are not fully understood. Understanding the molecular mechanisms underlying the response of GBM to chemotherapy is paramount for developing improved cancer therapeutics. In this study, we demonstrated that the loss of DOC-2/DAB2 interacting protein (DAB2IP) is responsible for TMZ-resistance in GBM through ATG9B. DAB2IP sensitized GBM to TMZ and suppressed TMZ-induced autophagy by negatively regulating ATG9B expression. A higher level of ATG9B expression was associated with GBM compared to low-grade glioma. The knockdown of ATG9B expression in GBM cells suppressed TMZ-induced autophagy as well as TMZ-resistance. Furthermore, we showed that DAB2IP negatively regulated ATG9B expression by blocking the Wnt/ß-catenin pathway. To enhance the benefit of TMZ and avoid therapeutic resistance, effective combination strategies were tested using a small molecule inhibitor blocking the Wnt/ß-catenin pathway in addition to TMZ. The combination treatment synergistically enhanced the efficacy of TMZ in GBM cells. In conclusion, the present study identified the mechanisms of TMZ-resistance of GBM mediated by DAB2IP and ATG9B which provides insight into a potential strategy to overcome TMZ chemo-resistance.

4-Dimethylaminopyridine-Catalyzed Metal-Free Aerobic Oxidation of Aryl α-Halo Esters to Aryl α-Keto Esters.

A novel, metal-free aerobic oxidation method is described. 4-Dimethylaminopyridine (DMAP) successfully catalyzed the oxidation of aryl α-halo esters to corresponding aryl α-keto esters (up to 95% yield) under mild reaction conditions (Li2CO3, dimethylacetamide, air, and room temperature). A mechanism has been proposed where the oxidation proceeds through a [3 + 2] cycloaddition between O2 in an air atmosphere and pyridinium ylides. The ylides are supposedly generated from aryl α-halo esters and DMAP in the presence of carbonates. Based on the plausible mechanism, the potential of DMAP as a catalyst in oxidation reactions was extended.