Repulsive Sema3E-Plexin-D1 signaling coordinates both axonal extension and steering via activating an autoregulatory factor, Mtss1.

Axon guidance molecules are critical for neuronal pathfinding because they regulate directionality and growth pace during nervous system development. However, the molecular mechanisms coordinating proper axonal extension and turning are poorly understood. Here, metastasis suppressor 1 (Mtss1), a membrane protrusion protein, ensured axonal extension while sensitizing axons to the Semaphorin 3E (Sema3E)-Plexin-D1 repulsive cue. Sema3E-Plexin-D1 signaling enhanced Mtss1 expression in projecting striatonigral neurons. Mtss1 localized to the neurite axonal side and regulated neurite outgrowth in cultured neurons. Mtss1 also aided Plexin-D1 trafficking to the growth cone, where it signaled a repulsive cue to Sema3E. Mtss1 ablation reduced neurite extension and growth cone collapse in cultured neurons. Mtss1-knockout mice exhibited fewer striatonigral projections and irregular axonal routes, and these defects were recapitulated in Plxnd1- or Sema3e-knockout mice. These findings demonstrate that repulsive axon guidance activates an exquisite autoregulatory program coordinating both axonal extension and steering during neuronal pathfinding.

EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.

The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.

Low-Dose Ionizing Radiation-Crosslinking Immunoprecipitation (LDIR-CLIP) Identified Irradiation-Sensitive RNAs for RNA-Binding Protein HuR-Mediated Decay.

Although ionizing radiation (IR) is widely used for therapeutic and research purposes, studies on low-dose ionizing radiation (LDIR) are limited compared with those on other IR approaches, such as high-dose gamma irradiation and ultraviolet irradiation. High-dose IR affects DNA damage response and nucleotide-protein crosslinking, among other processes; however, the molecular consequences of LDIR have been poorly investigated. Here, we developed a method to profile RNA species crosslinked to an RNA-binding protein, namely, human antigen R (HuR), using LDIR and high-throughput RNA sequencing. The RNA fragments isolated via LDIR-crosslinking and immunoprecipitation sequencing were crosslinked to HuR and protected from RNase-mediated digestion. Upon crosslinking HuR to target mRNAs such as PAX6, ZFP91, NR2F6, and CAND2, the transcripts degraded rapidly in human cell lines. Additionally, PAX6 and NR2F6 downregulation mediated the beneficial effects of LDIR on cell viability. Thus, our approach provides a method for investigating post-transcriptional gene regulation using LDIR.

Multiple toll-like receptors (TLRs) display differential bacterial and ligand specificity in the earthworm, Eisenia andrei.

Toll-like receptors (TLRs), an ancient and well-conserved group of pattern recognition receptors (PRRs), recognize conserved pathogen-associated molecular patterns. TLRs consist of three domains: the extracellular N-terminal domain, containing one or more leucine-rich repeats (LRRs), responsible for the recognizing and binding of antigens; the type-I transmembrane domain; and the intracellular domain known as the Toll/Interleukin-1 receptor (TIR) domain required for the downstream signaling pathway. We identified six new full-length complementary DNA (cDNA) sequences, Ean-TLR1/2/3/4/5/6. The deduced amino acid sequences indicate that Ean-TLRs consist of one signal peptide, one LRR N-terminal domain (Ean-TLR4/5), varying numbers of LRRs, one (Ean-TLR1/2/3/4/5) or two (Ean-TLR6) LRR C-terminal domains, one type-I transmembrane domain, and a TIR domain. In addition, a TIR domain alignment revealed that three conserved motifs, designated as Box 1, Box 2, and Box 3, contain essential amino acid residues for downstream signaling activity. Phylogenetic analysis of earthworm TLRs generated two separate evolutionary branches representing single (sccTLR) and multiple (mccTLR) cysteine cluster TLRs. Ean-TLR1/2/3/4 (sccTLR type) and Ean-TLR6 (mccTLR type) were clustered with corresponding types of previously reported earthworm TLRs as well as TLRs from Clitellata and Polychaete. As PRRs, earthworm TLRs should be capable of sensing a diverse range of pathogens. Except for Ean-TLR3, which was not responsive to any bacteria, earthworm TLR expression was significantly induced by Gram-positive but not Gram-negative bacteria. Moreover, it is likely that earthworms can differentiate between different species of Gram-positive bacteria via their TLR responses. The ligand specificity of earthworm TLRs suggests that their pathogenic ligand recognition is likely to be as specific and diverse as the mammalian TLR pathogen-sensing system.

Microtubule acetylation induced by oxidative stress regulates subcellular distribution of lysosomal vesicles for amyloid-beta secretion.

Excessive production and accumulation of amyloid-beta (Aß) in the brain are one of the hallmarks of Alzheimer's disease (AD). Although oxidative stress is known to trigger and promote the progression of AD, the molecular relationship between oxidative stress and Aß production is not yet fully understood. In this study, we demonstrate that microtubule acetylation induced by oxidative stress plays a critical role in Aß production and secretion by altering the subcellular distribution of Aß precursor protein (APP)-containing lysosomal vesicles. Under oxidative stress, both H4-APPSwe/Ind and HEK293T-APPSwe/Ind cell lines showed increased microtubule acetylation and Aß secretion. Knockdown (KD) of alpha-tubulin N-acetyltransferase 1 (ATAT1) by using a lentiviral shRNA not only inhibited the generation of intermediate APP fragments, such as ß-CTF and AICD, but also suppressed Aß secretion. Oxidative stress promoted the dispersion of LAMP1-positive vesicles to the periphery of the cell through microtubule acetylation, leading to the formation of neutralized lysosomal vesicles (NLVs), which was inhibited by ATAT1 KD. Treatment of the cells with the dynein ATPase inhibitor EHNA or downregulation of LIS1, a regulator of dynein-mediated intracellular transport, increased the peripheral localization of NLVs and promoted Aß secretion, whereas KD of ADP ribosylation factor like GTPase 8B showed the opposite result. ATAT1 KD in the hippocampal region of the 5×FAD AD mouse model also showed significant reductions in Aß plaque accumulation and memory loss. Taken together, these findings suggest that oxidative stress-induced microtubule acetylation promotes the peripheral localization of lysosomal vesicles to form NLVs, thereby enhancing Aß secretion.

Downregulation of AP1S1 causes the lysosomal degradation of EGFR in non-small cell lung cancer.

Matrix stiffness has been shown to play a critical role in cancer progression by influencing various cellular processes, including epidermal growth factor (EGF) signaling. However, the underlying molecular mechanisms are not fully understood. Here, we investigated the role of adaptor-related protein complex 1 subunit sigma 1 (AP1S1), a component of adaptor protein complex-1, in the regulation of EGF receptor (EGFR) intracellular trafficking during cancer cell progression. We found that AP1S1 expression was upregulated under stiff matrix conditions, resulting in the regulation of EGFR trafficking in non-small cell lung adenocarcinoma cells. Knockout of AP1S1 caused the lysosomal degradation of EGFR, leading to suppressed EGF-induced anaplastic lymphoma receptor tyrosine kinase phosphorylation. In addition, the downregulation of AP1S1 increased the sensitivity of H1975 cancer cells, which are resistant to tyrosine kinase inhibitors, to erlotinib. Collectively, our results suggest that AP1S1 could regulate EGFR recycling under stiff matrix conditions, and AP1S1 inhibition could be a novel strategy for treating cancer cells resistant to EGFR-targeted anticancer drugs.

Disruption of type I interferon pathway and reduced production of IFN-α by parabens in virus-infected dendritic cells.

BACKGROUND: Parabens are widely used preservatives commonly found in foods, cosmetics, and industrial products. Several studies have examined the effects of parabens on human health owing to widespread and continuous exposure to them in daily life. However, little is known about their immune-regulatory effects. OBJECTIVE: Here, we aimed to investigate whether methylparaben, ethylparaben, and propylparaben affect the function of dendritic cells (DCs) as the most potent antigen-presenting cells that play a critical role in the initiation of adaptive immune responses. METHODS: Bone-marrow derived DCs (BMDCs) were treated with three types of parabens (methylparaben, ethylparaben, and propylparaben) for 12 h. Subsequently, the transcriptomic profile was analyzed using RNA sequencing with further gene set enrichment analysis based on commonly regulated differentially expressed genes (DEGs). To test whether parabens suppress the production of type-I interferons (IFN-I) in BMDCs during viral infection, BMDCs or paraben-treated BMDCs were infected with Lymphocytic Choriomeningitis Virus (LCMV) at 10 multiplicity of infection (MOI) and measured the production of IFN-α1. RESULTS: Transcriptomic analyses revealed that all three types of parabens reduced the transcription levels of genes in virus infection-associated pathways, such as IFN-I responses in BMDCs. Furthermore, parabens considerably reduced IFN-α1 production in the virus-infected BMDCs. CONCLUSION: Our study is the first to show that parabens may modulate anti-viral immune responses by regulating DCs.

Slit-Robo expression in the leech nervous system: insights into eyespot evolution.

BACKGROUND: Slit and Robo are evolutionarily conserved ligand and receptor proteins, respectively, but the number of slit and robo gene paralogs varies across recent bilaterian genomes. Previous studies indicate that this ligand-receptor complex is involved in axon guidance. Given the lack of data regarding Slit/Robo in the Lophotrochozoa compared to Ecdysozoa and Deuterostomia, the present study aims to identify and characterize the expression of Slit/Robo orthologs in leech development. RESULTS: We identified one slit (Hau-slit), and two robo genes (Hau-robo1 and Hau-robo2), and characterized their expression spatiotemporally during the development of the glossiphoniid leech Helobdella austinensis. Throughout segmentation and organogenesis, Hau-slit and Hau-robo1 are broadly expressed in complex and roughly complementary patterns in the ventral and dorsal midline, nerve ganglia, foregut, visceral mesoderm and/or endoderm of the crop, rectum and reproductive organs. Before yolk exhaustion, Hau-robo1 is also expressed where the pigmented eye spots will later develop, and Hau-slit is expressed in the area between these future eye spots. In contrast, Hau-robo2 expression is extremely limited, appearing first in the developing pigmented eye spots, and later in the three additional pairs of cryptic eye spots in head region that never develop pigment. Comparing the expression of robo orthologs between H. austinensis and another glossiphoniid leech, Alboglossiphonia lata allows to that robo1 and robo2 operate combinatorially to differentially specify pigmented and cryptic eyespots within the glossiphoniid leeches. CONCLUSIONS: Our results support a conserved role in neurogenesis, midline formation and eye spot development for Slit/Robo in the Lophotrochozoa, and provide relevant data for evo-devo studies related to nervous system evolution.

Polypropylene microplastics promote metastatic features in human breast cancer.

Microplastics (MPs) are now a global issue due to increased plastic production and use. Recently, various studies have been performed in response to the human health risk assessment. However, these studies have focused on spherical MPs, which have smooth edges and a spherical shape and account for less than 1% of MPs in nature. Unfortunately, studies on fragment-type MPs are very limited and remain in the initial stages. In this study, we studied the effect that 16.4 µm fragment type polypropylene (PP) MPs, which have an irregular shape and sharp edges and form naturally in the environment, had on breast cancer. The detrimental effects of PPMPs on breast cancer metastasis were examined. Here, 1.6 mg/ml of PPMP, which does not induce cytotoxicity in MDA-MB-231, was used, and at this concentration, PPMP did not induce morphological changes or cellular migrating in the MDA-MB-231 and MCF-7 cells. However, PPMP incubation for 24 hours in the MDA-MB-231 cells significantly altered the level of cell cycle-related transcripts in an RNA-seq analysis. When confirmed by qRT-PCR, the gene expression of TMBIM6, AP2M1, and PTP4A2 was increased, while the transcript level of FTH1 was decreased. Further, secretion of the pro-inflammatory cytokine IL-6 from cancer cells was elevated with the incubation of PPMP for 12 hours. These results suggest that PPMP enhances metastasis-related gene expression and cytokines in breast cancer cells, exacerbating breast cancer metastasis.

KPNA3 promotes epithelial-mesenchymal transition by regulating TGF-ß and AKT signaling pathways in MDA-MB-231, a triple-negative breast cancer cell line.

Karyopherin-α3 (KPNA3), a karyopherin- α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMTrelated genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-ß or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-ß-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type. [BMB Reports 2023; 56(2): 120-125].

c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor that regulates early retinal development.

BACKGROUND: The transcription factor orthodenticle homeobox 2 (OTX2) has critical functions in brain and eye development, and its mutations in humans are related to retinal diseases, such as ocular coloboma and microphthalmia. However, the regulatory mechanisms of OTX2 are poorly identified. OBJECTIVE: The identification of JNK1 as an OTX2 regulatory protein through the protein interaction and phosphorylation. METHODS: To identify the binding partner of OTX2, we performed co-immunoprecipitation and detected with a pooled antibody that targeted effective kinases. The protein interaction between JNK1 and OTX2 was identified with the co-immunoprecipitation and immunocytochemistry. In vivo and in vitro kinase assay of JNK1 was performed to detect the phosphorylation of OTX2 by JNK1. RESULTS: JNK1 directly interacted with OTX2 through the transactivation domain at the c-terminal region. The protein-protein interaction and co-localization between JNK1 and OTX2 were further validated in the developing P0 mouse retina. In addition, we confirmed that the inactivation of JNK1 K55N mutant significantly reduced the JNK1-mediated phosphorylation of OTX2 by performing an immune complex protein kinase assay. CONCLUSION: c-Jun N-terminal kinase 1 (JNK1) phosphorylates OTX2 transcription factor through the protein-protein interaction.

Hyaluronic Acid Nanoparticles as a Topical Agent for Treating Psoriasis.

Although conventional topical approaches for treating psoriasis have been offered as an alternative, there are still unmet medical needs such as low skin-penetrating efficacy and off-target adverse effects. A hyaluronic acid nanoparticle (HA-NP) formed by self-assembly of HA-hydrophobic moiety conjugates has been broadly studied as a nanocarrier for long-term and target-specific delivery of drugs, owing to their excellent physicochemical and biological characteristics. Here, we identify HA-NPs as topical therapeutics for treating psoriasis using in vivo skin penetration studies and psoriasis animal models. Transcutaneously administered HA-NPs were found to be accumulated and associated with pro-inflammatory macrophages in the inflamed dermis of a psoriasis mouse model. Importantly, HA-NP exerted potent therapeutic efficacy against psoriasis-like skin dermatitis in a size-dependent manner by suppressing innate immune responses and restoring skin barrier function without overt toxicity signs. The therapeutic efficacy of HA-NPs on psoriasis-like skin dermatitis was due to the outermost hydrophilic HA shell layer of HA-NPs, independent of the molecular weight of HA and hydrophobic moiety, and comparable with that of other conventional psoriasis therapeutics widely used in the clinical settings. Overall, HA-NPs have the potential as a topical nanomedicine for treating psoriasis effectively and safely.

Anti-influenza A virus activity by Agrimonia pilosa and Galla rhois extract mixture.

Influenza A virus (IAV) continues to threaten human health. To date, two classes of antiviral drugs have been approved to treat IAV infection, but the continuous emergence of the drug-resistant IAV mutant reinforces the need to develop new antiviral drugs. In this study, we aimed to investigate the anti-IAV activity of an aqueous mixture of Agrimonia pilosa and Galla rhois extracts (APRG64). We demonstrated that APRG64 significantly reduced the IAV-induced cytopathic effect, the transcription/expression of viral proteins, and the production of infectious viral particles. Among nine major components of APRG64, apigenin was identified as the main ingredient responsible for the anti-IAV activity. Interestingly, APRG64 and apigenin inhibited the cell attachment and entry of virus and polymerase activity. Importantly, intranasal administration of APRG64 or apigenin strongly reduced viral loads in the lungs of IAV-infected mice. Furthermore, oral administration of APRG64 significantly reduced the level of viral RNAs and the expression level of pro-inflammatory cytokines in the lungs, which protected mice from IAV-induced mortality. In conclusion, APRG64 could be an attractive antiviral drug to treat IAV infection.

The Kleisin Subunits of Cohesin are Involved in the Fate Determination of Embryonic Stem Cells.

As a potential candidate to generate an everlasting cell source to treat various diseases, embryonic stem cells are regarded as a promising therapeutic tool in the regenerative medicine field. Cohesin, a multi-functional complex that controls various cellular activities, plays roles not only in organizing chromosome dynamics but also in controlling transcriptional activities related to self-renewal and differentiation of stem cells. Here, we report a novel role of the α-kleisin subunits of cohesin (RAD21 and REC8) in the maintenance of the balance between these two stem-cell processes. By knocking down REC8, RAD21, or the non-kleisin cohesin subunit SMC3 in mouse embryonic stem cells, we show that reduction in cohesin level impairs their self-renewal. Interestingly, the transcriptomic analysis revealed that knocking down each cohesin subunit enables the differentiation of embryonic stem cells into specific lineages. Specifically, embryonic stem cells in which cohesin subunit RAD21 were knocked down differentiated into cells expressing neural alongside germline lineage markers. Thus, we conclude that cohesin appears to control the fate determination of embryonic stem cells.

Peptidoglycan recognition proteins from the earthworm, Eisenia andrei: Differential inducibility and tissue-specific expression.

Several pattern recognition receptors (PRRs) involved in innate immunity have been identified and characterized in earthworms. Peptidoglycan recognition proteins (PGRPs) are highly conserved PRRs that activate effector pathways such as prophenoloxidase cascade and Toll-like receptor pathway. In addition, PGRPs function as an enzyme, N-acetylmuramoyl-l-alanine amidase (NAMLAA), to directly hydrolyze peptidoglycan. We identified four full-length complementary DNA (cDNA) sequences, Ean-PGRP1/2/3/4, in Eisenia andrei, an earthworm. Sequence and phylogenetic analyses indicate that earthworm PGRP orthologs resemble short PGRP member proteins. The subcellular localizations of four Ean-PGRPs lacking the transmembrane domain are predicted to be extracellular or cytoplasmic. All Ean-PGRPs contain a highly conserved PGRP domain with a conserved Zn2+ binding site including a tyrosine residue essential for active amidase activity. Three highly conserved amino-acid residues (His, Trp, and Thr) necessary for amidase activity are also present, indicating that the Ean-PGRPs can be predicted to have amidase activity. Furthermore, we demonstrate that the Ean-PGRP genes are differentially induced by certain bacterial species, suggesting that the innate immune system of earthworms is likely to be somewhat specific rather than entirely non-specific. Tissue expression patterns indicate that Ean-PGRP mRNAs are primarily expressed in the immune-competent tissues and that their expression is tissue-specific according to Ean-PGRP types, particularly for Ean-PGRP1.

Multiomics analyses reveal early metabolic imbalance and mitochondrial stress in neonatal photoreceptors leading to cell death in Pde6brd1/rd1 mouse model of retinal degeneration.

Retinal diseases exhibit extensive genetic heterogeneity and complex etiology with varying onset and severity. Mutations in over 200 genes can lead to photoreceptor dysfunction and/or cell death in retinal neurodegeneration. To deduce molecular pathways that initiate and/or drive cell death, we adopted a temporal multiomics approach and examined molecular and cellular events in newborn and developing photoreceptors before the onset of degeneration in a widely-used Pde6brd1/rd1 (rd1) mouse, a model of autosomal recessive retinitis pigmentosa caused by PDE6B mutations. Transcriptome profiling of neonatal and developing rods from the rd1 retina revealed early downregulation of genes associated with anabolic pathways and energy metabolism. Quantitative proteomics of rd1 retina showed early changes in calcium signaling and oxidative phosphorylation, with specific partial bypass of complex I electron transfer, which precede the onset of cell death. Concurrently, we detected alterations in central carbon metabolism, including dysregulation of components associated with glycolysis, pentose phosphate and purine biosynthesis. Ex vivo assays of oxygen consumption and transmission electron microscopy validated early and progressive mitochondrial stress and abnormalities in mitochondrial structure and function of rd1 rods. These data uncover mitochondrial overactivation and related metabolic alterations as determinants of early pathology and implicate aberrant calcium signaling as an initiator of higher mitochondrial stress. Our studies thus provide a mechanistic framework with mitochondrial damage and metabolic disruptions as early drivers of photoreceptor cell death in retinal degeneration.

Anti-inflammatory effect of Ailanthus altissima (Mill.) Swingle leaves in lipopolysaccharide-stimulated astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Activated astrocytes are involved in the progression of neurodegenerative diseases. Traditionally, Ailanthus altissima (Mill.) Swingle, widely distributed in East Asia, has been used as a medicine for the treatment of fever, gastric diseases, and inflammation. Although A. altissima has been reported to play an anti-inflammatory role in peripheral tissues or cells, its role in the central nervous system (CNS) remains unclear. AIM OF THE STUDY: In the present study, we investigated the anti-inflammatory effects and mechanism of action of A. altissima in primary astrocytes stimulated by lipopolysaccharide (LPS). MATERIALS AND METHODS: A nitrite assay was used to measure nitric oxide (NO) production, and the tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was performed to determine cytotoxicity. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and mitogen-activated protein kinase (MAPK) were determined with western blotting. Reverse-transcription PCR was used to assess the expression of inflammatory cytokines. The levels of reactive oxygen species were measured using 2,7-dichlorodihydrofluorescein diacetate. Luciferase assay and immunocytochemistry were used for assessing nuclear factor-kappa B (NF-κB) transcription and p65 localization, respectively. Memory and social interaction were analyzed using the Y-maze and three-chamber tests, respectively. RESULTS: The ethanol extract of A. altissima leaves (AAE) inhibited iNOS and COX-2 expression in LPS-stimulated astrocytes. Moreover, AAE reduced the transcription of various proinflammatory mediators, hindered NF-κB activation, and suppressed extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activation without p38 activation. Ultra-high performance liquid chromatography with mass spectrometry analysis revealed that AAE comprised ethyl gallate, quercetin, and kaempferol, along with luteolin, which has anti-inflammatory properties, and repressed LPS-induced nitrite levels and the nuclear translocation of p65. Finally, oral administration of AAE attenuated LPS-induced memory and social impairment in mice and repressed LPS-induced ERK and JNK activation in the cortices of mice. CONCLUSION: AAE could have therapeutic uses in the treatment of neuroinflammatory diseases via suppression of astrocyte activation.

Beta-Pix-dynamin 2 complex promotes colorectal cancer progression by facilitating membrane dynamics.

PURPOSE: Spatiotemporal regulation of cell membrane dynamics is a major process that promotes cancer cell invasion by acting as a driving force for cell migration. Beta-Pix (ßPix), a guanine nucleotide exchange factor for Rac1, has been reported to be involved in actin-mediated cellular processes, such as cell migration, by interacting with various proteins. As yet, however, the molecular mechanisms underlying ßPix-mediated cancer cell invasion remain unclear. METHODS: The clinical significance of ßPix was analyzed in patients with colorectal cancer (CRC) using public clinical databases. Pull-down and immunoprecipitation assays were employed to identify novel binding partners for ßPix. Additionally, various cell biological assays including immunocytochemistry and time-lapse video microscopy were performed to assess the effects of ßPix on CRC progression. A ßPix-SH3 antibody delivery system was used to determine the effects of the ßPix-Dyn2 complex in CRC cells. RESULTS: We found that the Src homology 3 (SH3) domain of ßPix interacts with the proline-rich domain of Dynamin 2 (Dyn2), a large GTPase. The ßPix-Dyn2 interaction promoted lamellipodia formation, along with plasma membrane localization of membrane-type 1 matrix metalloproteinase (MT1-MMP). Furthermore, we found that Src kinase-mediated phosphorylation of the tyrosine residue at position 442 of ßPix enhanced ßPix-Dyn2 complex formation. Disruption of the ßPix-Dyn2 complex by ßPix-SH3 antibodies targeting intracellular ßPix inhibited CRC cell invasion. CONCLUSIONS: Our data indicate that spatiotemporal regulation of the Src-ßPix-Dyn2 axis is crucial for CRC cell invasion by promoting membrane dynamics and MT1-MMP recruitment into the leading edge. The development of inhibitors that disrupt the ßPix-Dyn2 complex may be a useful therapeutic strategy for CRC.

A Fully Human Monoclonal Antibody Targeting cKIT Is a Potent Inhibitor of Pathological Choroidal Neovascularization in Mice.

Stem cell factor (SCF) and its receptor, cKIT, are novel regulators of pathological neovascularization in the eye, which suggests that inhibition of SCF/cKIT signaling may be a novel pharmacological strategy for treating neovascular age-related macular degeneration (AMD). This study evaluated the therapeutic potential of a newly developed fully human monoclonal antibody targeting cKIT, NN2101, in a murine model of neovascular AMD. In hypoxic human endothelial cells, NN2101 substantially inhibited the SCF-induced increase in angiogenesis and activation of the cKIT signaling pathway. In a murine model of neovascular AMD, intravitreal injection of NN2101 substantially inhibited the SCF/cKIT-mediated choroidal neovascularization (CNV), with efficacy comparable to aflibercept, a vascular endothelial growth factor inhibitor. A combined intravitreal injection of NN2101 and aflibercept resulted in an additive therapeutic effect on CNV. NN2101 neither caused ocular toxicity nor interfered with the early retinal vascular development in mice. Ocular pharmacokinetic analysis in rabbits indicated that NN2101 demonstrated a pharmacokinetic profile suitable for intravitreal injection. These findings provide the first evidence of the potential use of the anti-cKIT blocking antibody, NN2101, as an alternative or additive therapeutic for the treatment of neovascular AMD.

Proneural genes define ground-state rules to regulate neurogenic patterning and cortical folding.

Asymmetric neuronal expansion is thought to drive evolutionary transitions between lissencephalic and gyrencephalic cerebral cortices. We report that Neurog2 and Ascl1 proneural genes together sustain neurogenic continuity and lissencephaly in rodent cortices. Using transgenic reporter mice and human cerebral organoids, we found that Neurog2 and Ascl1 expression defines a continuum of four lineage-biased neural progenitor cell (NPC) pools. Double+ NPCs, at the hierarchical apex, are least lineage restricted due to Neurog2-Ascl1 cross-repression and display unique features of multipotency (more open chromatin, complex gene regulatory network, G2 pausing). Strikingly, selectively eliminating double+ NPCs by crossing Neurog2-Ascl1 split-Cre mice with diphtheria toxin-dependent "deleter" strains locally disrupts Notch signaling, perturbs neurogenic symmetry, and triggers cortical folding. In support of our discovery that double+ NPCs are Notch-ligand-expressing "niche" cells that control neurogenic periodicity and cortical folding, NEUROG2, ASCL1, and HES1 transcript distribution is modular (adjacent high/low zones) in gyrencephalic macaque cortices, prefiguring future folds.