Brain-specific Angiogenesis Inhibitor Research Articles

Emerging Roles of BAI Adhesion-GPCRs in Synapse Development and Plasticity.

Synapses mediate communication between neurons and enable the brain to change in response to experience, which is essential for learning and memory. The sites of most excitatory synapses in the brain, dendritic spines, undergo rapid remodeling that is important for neural circuit formation and synaptic plasticity. Abnormalities in synapse and spine formation and plasticity are associated with a broad range of brain disorders, including intellectual disabilities, autism spectrum disorders (ASD), and schizophrenia. Thus, elucidating the mechanisms that regulate these neuronal processes is critical for understanding brain function and disease. The brain-specific angiogenesis inhibitor (BAI) subfamily of adhesion G-protein-coupled receptors (adhesion-GPCRs) has recently emerged as central regulators of synapse development and plasticity. In this review, we will summarize the current knowledge regarding the roles of BAIs at synapses, highlighting their regulation, downstream signaling, and physiological functions, while noting the roles of other adhesion-GPCRs at synapses. We will also discuss the relevance of BAIs in various neurological and psychiatric disorders and consider their potential importance as pharmacological targets in the treatment of these diseases.

Expression of brain‑specific angiogenesis inhibitor‑1 and association with p53, microvessel density and vascular endothelial growth factor in the tissue of human bladder transitional cell carcinoma.

The aim of the present study was to investigate the expression levels of brain‑specific angiogenesis inhibitor‑1 (BAI‑1) in bladder transitional cell carcinoma (BTCC) at different stages and the mechanism by which it inhibits tumor endothelial cell proliferation. Normal bladder mucosa biopsy specimens were obtained as the control group, and human BTCC biopsy specimens were used as the study group. Immunohistochemical assays were used to detect the expression levels of BAI‑1, vascular endothelial growth factor (VEGF) and mutant p53, in addition to microvessel density (MVD) in the tissues. Western blotting was used to analyze the differential expression of BAI‑1 in the two samples. Statistical analysis was performed, which indicated that BAI‑1 expression levels in the normal bladder mucosa group were significantly higher than those in the BTCC group and were associated with clinical staging. BAI‑1 levels in the T1 stage BTCC tissues were higher than those in the T2‑4 stage BTCC tissues (P<0.05). BAI‑1 expression levels were negatively correlated with those of VEGF (r=‑0.661, P<0.001), mutant p53 (r=‑0.406, P=0.002) and with the MVD (r=‑0.675, P<0.001). BAI‑1 may be involved in the negative regulation of BTCC microvascular proliferation, and its expression may be associated with a reduction in p53 mutations.

Apoptotic cells trigger a membrane-initiated pathway to increase ABCA1.

Macrophages clear millions of apoptotic cells daily and, during this process, take up large quantities of cholesterol. The membrane transporter ABCA1 is a key player in cholesterol efflux from macrophages and has been shown via human genetic studies to provide protection against cardiovascular disease. How the apoptotic cell clearance process is linked to macrophage ABCA1 expression is not known. Here, we identified a plasma membrane-initiated signaling pathway that drives a rapid upregulation of ABCA1 mRNA and protein. This pathway involves the phagocytic receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recognizes phosphatidylserine on apoptotic cells, and the intracellular signaling intermediates engulfment cell motility 1 (ELMO1) and Rac1, as ABCA1 induction was attenuated in primary macrophages from mice lacking these molecules. Moreover, this apoptotic cell-initiated pathway functioned independently of the liver X receptor (LXR) sterol-sensing machinery that is known to regulate ABCA1 expression and cholesterol efflux. When placed on a high-fat diet, mice lacking BAI1 had increased numbers of apoptotic cells in their aortic roots, which correlated with altered lipid profiles. In contrast, macrophages from engineered mice with transgenic BAI1 overexpression showed greater ABCA1 induction in response to apoptotic cells compared with those from control animals. Collectively, these data identify a membrane-initiated pathway that is triggered by apoptotic cells to enhance ABCA1 within engulfing phagocytes and with functional consequences in vivo.

BAI1 regulates spatial learning and synaptic plasticity in the hippocampus.

Synaptic plasticity is the ability of synapses to modulate the strength of neuronal connections; however, the molecular factors that regulate this feature are incompletely understood. Here, we demonstrated that mice lacking brain-specific angiogenesis inhibitor 1 (BAI1) have severe deficits in hippocampus-dependent spatial learning and memory that are accompanied by enhanced long-term potentiation (LTP), impaired long-term depression (LTD), and a thinning of the postsynaptic density (PSD) at hippocampal synapses. We showed that compared with WT animals, mice lacking Bai1 exhibit reduced protein levels of the canonical PSD component PSD-95 in the brain, which stems from protein destabilization. We determined that BAI1 prevents PSD-95 polyubiquitination and degradation through an interaction with murine double minute 2 (MDM2), the E3 ubiquitin ligase that regulates PSD-95 stability. Restoration of PSD-95 expression in hippocampal neurons in BAI1-deficient mice by viral gene therapy was sufficient to compensate for Bai1 loss and rescued deficits in synaptic plasticity. Together, our results reveal that interaction of BAI1 with MDM2 in the brain modulates PSD-95 levels and thereby regulates synaptic plasticity. Moreover, these results suggest that targeting this pathway has therapeutic potential for a variety of neurological disorders.

Structures of C1q-like Proteins Reveal Unique Features among the C1q/TNF Superfamily

C1q-like (C1QL) -1, -2, and -3 proteins are encoded by homologous genes that are highly expressed in brain. C1QLs bind to brain-specific angiogenesis inhibitor 3 (BAI3), an adhesion-type G-protein coupled receptor that may regulate dendritic morphology by organizing actin filaments. To begin to understand the function of C1QLs, we determined high-resolution crystal structures of the globular C1q-domains of C1QL1, C1QL2, and C1QL3. Each structure is a trimer, with each protomer forming a jelly-roll fold consisting of 10 β strands. Moreover, C1QL trimers may assemble into higher-order oligomers similar to adiponectin and contain four Ca(2+)-binding sites along the trimeric symmetry axis, as well as additional surface Ca(2+)-binding sites. Mutation of Ca(2+)-coordinating residues along the trimeric symmetry axis lowered the Ca(2+)-binding affinity and protein stability. Our results reveal unique structural features of C1QLs among C1q/TNF superfamily proteins that may be associated with their specific brain functions.

Identification of biomarkers for metastatic osteosarcoma based on DNA microarray data.

Osteosarcoma (OS) is amalignant bone tumor very often with pulmonary metastasis and is the main cause of OS mortality. The objective of this study was to screen for possible biomarkers of metastatic OS to explore the mechanisms of pulmonary metastasis of OS through network construction. GSE14359 was downloaded from the Gene Expression Omnibus database, which included 5 samples from conventional OS group with 2 replicates and 4 samples from OS pulmonary metastasis group in duplicate. Differentially expressed genes (DEGs) between two groups were identified by limma packages in Rand classical t-test with the threshold of the false discovery rate (FDR) <0.05. The Database for Annotation, Visualization and Integrated Discovery (DAVID) were then used to perform functional annotation (FDR < 0.01). Differential coexpression network was constructed with subspace differential coexpression analysis (SDC), and genes with high degrees in the differential coexpression network were identified. A total of 1344 genes were screened as DEGs, including 677 up- and 667 down-regulated DEGs in the pulmonary metastasis of OS. Thirty-one significantly enriched functions were obtained, such as blood vessel morphogenesis, defense response, cell death and so on. DEGs with high degrees (brain-specific angiogenesis inhibitor 2 (BAI2), formin-like 1 (FMNL1), dual-specificity phosphatase 7 (DUSP7), transient receptor potential melastatin 2 (TRPM2), CBP80/20-dependent translation initiation factor (KIAA0427) and C120rf35) in the differential coexpression network were found. BAI2, FMNL1, DUSP7 and TRPM2 may be useful markers for predicting tumor metastasis and therapeutic targets for the treatment of OS patients with metastasis.

Anterograde C1ql1 Signaling Is Required in Order to Determine and Maintain a Single-Winner Climbing Fiber in the Mouse Cerebellum

Neuronal networks are dynamically modified by selective synapse pruning during development and adulthood. However, how certain connections win the competition with others and are subsequently maintained is not fully understood. Here, we show that C1ql1, a member of the C1q family of proteins, is provided by climbing fibers (CFs) and serves as a crucial anterograde signal to determine and maintain the single-winner CF in the mouse cerebellum throughout development and adulthood. C1ql1 specifically binds to the brain-specific angiogenesis inhibitor 3 (Bai3), which is a member of the cell-adhesion G-protein-coupled receptor family and expressed on postsynaptic Purkinje cells. C1ql1-Bai3 signaling is required for motor learning but not for gross motor performance or coordination. Because related family members of C1ql1 and Bai3 are expressed in various brain regions, the mechanism described here likely applies to synapse formation, maintenance, and function in multiple neuronal circuits essential for important brain functions.

CS-38 * BAI1 IS A NEW TUMOR SUPPRESSOR IN MEDULLOBLASTOMA

Brain-specific Angiogenesis Inhibitor 1 (BAI1) is a seven transmembrane G protein-coupled receptor (GPCR) with potent anti-angiogenic and anti-tumorigenic properties in gliomas. Our novel findings show that BAI1 expression is significantly reduced in human medulloblastoma (MB) by epigenetic mechanisms, leading us to hypothesize that loss of expression of BAI1 may favor tumor development during cerebellar development. To test this hypothesis, we generated a Bai1 knockout (KO) mouse and detected aberrant activation of Sonic hedgehog signaling and a thicker external granular layer during early postnatal cerebellum development. Furthermore, upon crossing the Bai1 KO mice with mice heterozygous for patched 1 (ptc1 + /-), we found that deficiency of Bai1 dramatically accelerated MB tumorigenesis, the first demonstration that a reduction in Bai1 dosage can promote MB formation in vivo. Moreover, restoration of BAI1 expression in human MB cells inhibited cell growth both in vitro and in vivo. These results support that BAI1 is a novel tumor suppressor for MB and that down-regulation of BAI1 in the cerebellum may drive inappropriate downstream growth signaling and contribute to neoplastic transformation leading to the formation of MB. To further elucidate the molecular mechanisms, we are currently exploring the Bai1 downstream signaling involved in MB tumorigenesis and will present the related findings. Our results also suggest that restoration of BAI1 expression may represent a novel therapeutic intervention for MB. Specifically, we demonstrate that small molecule epigenetic inhibitors of either EZH2 or MBD2 can reactivate BAI1 expression. Taken together, our novel findings provide insight into the neurobiological mechanisms underlying MB tumorigenesis and provide the rationale for the use of epigenetic therapeutics against MB.

G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates

Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion.

BAIAP2 Is Related to Emotional Modulation of Human Memory Strength

Memory performance is the result of many distinct mental processes, such as memory encoding, forgetting, and modulation of memory strength by emotional arousal. These processes, which are subserved by partly distinct molecular profiles, are not always amenable to direct observation. Therefore, computational models can be used to make inferences about specific mental processes and to study their genetic underpinnings. Here we combined a computational model-based analysis of memory-related processes with high density genetic information derived from a genome-wide study in healthy young adults. After identifying the best-fitting model for a verbal memory task and estimating the best-fitting individual cognitive parameters, we found a common variant in the gene encoding the brain-specific angiogenesis inhibitor 1-associated protein 2 (BAIAP2) that was related to the model parameter reflecting modulation of verbal memory strength by negative valence. We also observed an association between the same genetic variant and a similar emotional modulation phenotype in a different population performing a picture memory task. Furthermore, using functional neuroimaging we found robust genotype-dependent differences in activity of the parahippocampal cortex that were specifically related to successful memory encoding of negative versus neutral information. Finally, we analyzed cortical gene expression data of 193 deceased subjects and detected significant BAIAP2 genotype-dependent differences in BAIAP2 mRNA levels. Our findings suggest that model-based dissociation of specific cognitive parameters can improve the understanding of genetic underpinnings of human learning and memory.

Baiap3 as a genetic marker associated with anxiety and benzodiazepine abuse in mouse and man

The brain specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated-13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis. Baiap3 has a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. We identify Baiap3 as a first genetic risk marker for anxiety and benzodiazepine use disorder in mice and humans. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, the latter being more pronounced in female than in male animals. We further identify human BAIAP3 risk genotypes that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we find that male, but not female Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices reveals an increase in basal network activity and an altered response to diazepam. Thus, Baiap3/BAIAP3 is the first gene shown to be gender-specifically associated with increased anxiety and benzodiazepine abuse in mice and humans. Further analysis of Baiap3/BAIAP3-related functions may therefore help elucidate mechanisms underlying the development of both disorders.

Brain-specific Angiogenesis Inhibitor-1 Signaling, Regulation, and Enrichment in the Postsynaptic Density

Brain-specific angiogenesis inhibitor-1 (BAI1) is an adhesion G protein-coupled receptor that has been studied primarily for its anti-angiogenic and anti-tumorigenic properties. We found that overexpression of BAI1 results in activation of the Rho pathway via a Gα(12/13)-dependent mechanism, with truncation of the BAI1 N terminus resulting in a dramatic enhancement in receptor signaling. This constitutive activity of the truncated BAI1 mutant also resulted in enhanced downstream phosphorylation of ERK as well as increased receptor association with β-arrestin2 and increased ubiquitination of the receptor. To gain insights into the regulation of BAI1 signaling, we screened the C terminus of BAI1 against a proteomic array of PDZ domains to identify novel interacting partners. These screens revealed that the BAI1 C terminus interacts with a variety of PDZ domains from synaptic proteins, including MAGI-3. Removal of the BAI1 PDZ-binding motif resulted in attenuation of receptor signaling to Rho but had no effect on ERK activation. Conversely, co-expression with MAGI-3 was found to potentiate signaling to ERK by constitutively active BAI1 in a manner that was dependent on the PDZ-binding motif of the receptor. Biochemical fractionation studies revealed that BAI1 is highly enriched in post-synaptic density fractions, a finding consistent with our observations that BAI1 can interact with PDZ proteins known to be concentrated in the post-synaptic density. These findings demonstrate that BAI1 is a synaptic receptor that can activate both the Rho and ERK pathways, with the N-terminal and C-terminal regions of the receptor playing key roles in the regulation of BAI1 signaling activity.

The integrin inhibitor cilengitide enhances the anti-glioma efficacy of vasculostatin-expressing oncolytic virus

Oncolytic viral (OV) therapy has been considered as a promising treatment modality for brain tumors. Vasculostatin, the fragment of brain-specific angiogenesis inhibitor-1, shows anti-angiogenic activity against malignant gliomas. Previously, a vasculostatin-expressing oncolytic herpes simplex virus-1, Rapid Antiangiogenesis Mediated By Oncolytic virus (RAMBO), was reported to have a potent antitumor effect. Here, we investigated the therapeutic efficacy of RAMBO and cilengitide, an integrin inhibitor, combination therapy for malignant glioma. In vitro, tube formation was significantly decreased in RAMBO and cilengitide combination treatment compared with RAMBO or cilengitide monotherapy. Moreover, combination treatment induced a synergistic suppressive effect on endothelial cell migration compared with the control virus. RAMBO, combined with cilengitide, induced synergistic cytotoxicity on glioma cells. In the caspase-8 and -9 assays, the relative absorption of U87ΔEGFR cell clusters treated with cilengitide and with RAMBO was significantly higher than that of those treated with control. In addition, the activity of caspase 3/7 was significantly increased with combination therapy. In vivo, there was a significant increase in the survival of mice treated with combination therapy compared with RAMBO or cilengitide monotherapy. These results indicate that cilengitide enhanced vasculostatin-expressing OV therapy for malignant glioma and provide a rationale for designing future clinical trials combining these two agents.

Establishment of metastatic molecule diagnostic model for hepatocellular carcinoma

Objective To construct the molecular model and screen the key molecules for evaluation of tumor metastatic potential in nude mice bearing hepatocellular carcinoma (HCC) xenografts.Methods Orthotopic HCC models were established by implantation of human HCC cell line MHCC97H xenografts with high metastatic potential.Thirty-six nude mice bearing HCC were randomized into three groups 14 days post-operation,including palliative resection group (samples A,B),sham operation group (sample C1),and control group (sample C2).Six mice in each group were sacrificed by cervical dislocation 14 days after palliative resection.Oligo Tumor Metastasis Microarray (OHS-028) and GEArray Expression Analysis Suite software were adopted for gene analysis.The methods of support vector machine (SVM),gene significance analysis and gene correlation degree analysis were used to find the markers that could differentiate the metastatic potential of HCC.Gene function net was constructed based on the special gene clustering analysis and multi-dimensional scale.The relevance vector machine (RVM) was used to deal with regression and classification problems.Real-time fluorescence quantitative polymerase chain reaction (FQ-PCR) was applied to detect mRNA expression.Results By the method of support vector machine (SVM),we found the markers composed of 12 genes (BAI1,MTA2,MTA1,SMAD2,GNRH1,CDHS,ITGB3,CHD4,GZMA,ITGA7,CXCR4,and TSHR) could differentiate the metastatic potential of HCC with positive predictive rate of 88.9％ and 93.5％ in the groups with high and low metastatic potential respectively.In addition,it was found that MTSS1,transforming growth factor-β1 (TGF-β1),SMAD2,interleukin-1 β (IL-1 β) and matrix metalloproteinase-7 (MMP-7) were situated in the central position of the gene function net with high metastatic potential,which was confirmed by Real-time PCR.Conclusion The constructed molecule diagnostic model and the key molecules may be used to evaluate metastatic potential of HCC. Key words: Carcinoma, hepatocellular ; Molecular model; Invasion ; Metastasis

The adhesion-GPCR BAI1 regulates synaptogenesis by controlling the recruitment of the Par3/Tiam1 polarity complex to synaptic sites.

Excitatory synapses are polarized structures that primarily reside on dendritic spines in the brain. The small GTPase Rac1 regulates the development and plasticity of synapses and spines by modulating actin dynamics. By restricting the Rac1-guanine nucleotide exchange factor Tiam1 to spines, the polarity protein Par3 promotes synapse development by spatially controlling Rac1 activation. However, the mechanism for recruiting Par3 to spines is unknown. Here, we identify brain-specific angiogenesis inhibitor 1 (BAI1) as a synaptic adhesion GPCR that is required for spinogenesis and synaptogenesis in mice and rats. We show that BAI1 interacts with Par3/Tiam1 and recruits these proteins to synaptic sites. BAI1 knockdown results in Par3/Tiam1 mislocalization and loss of activated Rac1 and filamentous actin from spines. Interestingly, BAI1 also mediates Rac-dependent engulfment in professional phagocytes through its interaction with a different Rac1-guanine nucleotide exchange factor module, ELMO/DOCK180. However, this interaction is dispensable for BAI1's role in synapse development because a BAI1 mutant that cannot interact with ELMO/DOCK180 rescues spine defects in BAI1-knockdown neurons, whereas a mutant that cannot interact with Par3/Tiam1 rescues neither spine defects nor Par3 localization. Further, overexpression of Tiam1 rescues BAI1 knockdown spine phenotypes. These results indicate that BAI1 plays an important role in synaptogenesis that is mechanistically distinct from its role in phagocytosis. Furthermore, our results provide the first example of a cell surface receptor that targets members of the PAR polarity complex to synapses.

Understanding protein‐protein interaction network: a case study with glutaminase interacting protein

Glutaminase interacting protein (GIP) is a 124 amino acid long protein containing single PDZ domain. This protein intersects a number of important biological pathways, of which many are unknown. Its involvement in cancer pathways makes it a good target for drug design based on the structure. We resolved the solution structures of both free GIP and GIP in complex with C‐terminal peptide analog of glutaminase L to shed light on the mechanism of binding with the goal of future development of a potential inhibitor for GIP. To understand more of GIP's function, interactions with two target peptides were investigated using different biophysical methods. One of the peptide had the C‐terminus of brain‐specific angiogenesis inhibitor 2 (BAI2) and the other one used had a consensus PDZ class I binding motif. Both of the peptides showed moderate binding affinity toward GIP with BAI2 peptide having comparatively higher affinity. Elucidating the mechanism of interactions for different target partners would help to lay out the network of function for GIP. This research was financially supported by USDA PECASE Presidential Early Career Award for Scientists and Engineers award 2003–35302‐12930, NSF Grant IBN‐0628064, USDA Grant 2011–65503‐20030 and NIH Grant DK082397 to Smita Mohanty.

Expression of brain-specific angiogenesis inhibitor 1 is inversely correlated with pathological grade, angiogenesis and peritumoral brain edema in human astrocytomas

As the most common intracranial malignant neoplasms, astrocytomas are characterized by high neovascularization and severe peritumoral brain edema (PTBE). Angiogenesis is a prerequisite for the growth of solid tumors, including astrocytoma, and brain-specific angiogenesis inhibitor 1 (BAI1) is a novel angiogenesis inhibitor. In the present study, the expression levels of BAI1, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were investigated using immunohistochemical methods in 90 human brain astrocytoma specimens of various pathological grades and in 11 normal human brain tissues. Vascular endothelial cells were stained for CD105 and the microvessel density (MVD) was assessed. The volume of astrocytoma and PTBE in each case was evaluated by magnetic resonance imaging (MRI). The results showed that BAI1 was highly expressed in the normal brain tissues, but that the expression decreased with the rising pathological grades of astrocytoma, MVD number and PTBE, indicating that BAI1 expression was inversely correlated with these factors. Furthermore, it was observed that the expression of VEGF and bFGF were inversely correlated with BAI1 expression in the human brain astrocytomas. These results indicate that the BAI1 gene may be used as a marker of decreased tumor progression and tumoral neovascularization, as well as PTBE.

Genetic Markers of a Munc13 Protein Family Member, BAIAP3, Are Gender Specifically Associated with Anxiety and Benzodiazepine Abuse in Mice and Humans

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein

BackgroundHuman Immunodeficiency Virus-1 (HIV-1) associated neurocognitive disorders (HANDs) are accompanied by significant morbidity, which persists despite the use of combined antiretroviral therapy (cART). While activated microglia play a role in pathogenesis, changes in their immune effector functions, including phagocytosis and proinflammatory signaling pathways, are not well understood. We have identified leucine-rich repeat kinase 2 (LRRK2) as a novel regulator of microglial phagocytosis and activation in an in vitro model of HANDs, and hypothesize that LRRK2 kinase inhibition will attenuate microglial activation during HANDs.MethodsWe treated BV-2 immortalized mouse microglia cells with the HIV-1 trans activator of transcription (Tat) protein in the absence or presence of LRRK2 kinase inhibitor (LRRK2i). We used Western blot, qRT-PCR, immunocytochemistry and latex bead engulfment assays to analyze LRRK2 protein levels, proinflammatory cytokine and phagocytosis receptor expression, LRRK2 cellular distribution and phagocytosis, respectively. Finally, we utilized ex vivo microfluidic chambers containing primary hippocampal neurons and BV-2 microglia cells to investigate microglial phagocytosis of neuronal axons.ResultsWe found that Tat-treatment of BV-2 cells induced kinase activity associated phosphorylation of serine 935 on LRRK2 and caused the formation of cytoplasmic LRRK2 inclusions. LRRK2i decreased Tat-induced phosphorylation of serine 935 on LRRK2 and inhibited the formation of Tat-induced cytoplasmic LRRK2 inclusions. LRRK2i also decreased Tat-induced process extension in BV-2 cells. Furthermore, LRRK2i attenuated Tat-induced cytokine expression and latex bead engulfment. We examined relevant cellular targets in microfluidic chambers and found that Tat-treated BV-2 microglia cells cleared axonal arbor and engulfed neuronal elements, whereas saline treated controls did not. LRRK2i was found to protect axons in the presence of Tat-activated microglia, as well as AnnexinV, a phosphatidylserine-binding protein. In addition, LRRK2i decreased brain-specific angiogenesis inhibitor 1 (BAI1) receptor expression on BV-2 cells after Tat-treatment, a key receptor in phosphatidylserine-mediated phagocytosis.ConclusionTaken together, these results implicate LRRK2 as a key player in microglial inflammation and, in particular, in the phagocytosis of neuronal elements. These studies show that LRRK2 kinase inhibition may prove an effective therapeutic strategy for HANDs, as well as other neuroinflammatory conditions.

Moonlighting osteoclasts as undertakers of apoptotic cells

Rapid clearance of apoptotic cells, frequently referred to as efferocytosis, is crucial for the maintenance of tissue homeostasis and the prevention of autoimmunity. The common model of apoptotic cell clearance involves a system of released “Find me” and exposed “Eat me” signals on apoptotic cells, detected and recognized by matching receptors on macrophages or dendritic cells (DC), referred to as the phagocytic synapse. Osteoclasts share the monocyte lineage with these professional mononuclear phagocytes, thus raising the question if, in addition to bone resorption, osteoclasts can act as scavengers for apoptotic cells. Our qPCR data clearly show that osteoclasts express most of the genes required for dying cell clearance at mRNA levels similar to or even higher than those observed in M1-macrophages, M2-macrophages or DC. Our microscopical analyses reveal that osteoclasts in fact can bind and/or engulf apoptotic cells in an essentially serum-independent fashion. Together with our data on the abundance of the respective mRNAs, these results identify the vitronectin receptor (integrin ανβ3)/milk fat globule-EGF factor 8 protein (MFG-E8) axis, the scavenger receptors (CD36, CD68 and class A macrophage scavenger receptor (SR-A)), the complement/complement receptor axis, the Mer/Tyro3/Protein S axis, and the phosphatidylserine (PS) receptor brain-specific angiogenesis inhibitor 1 (BAI1) as the most promising candidates to be involved in osteoclast-mediated efferocytosis.