ABSTRACT
Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl-/- mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.
Subject(s)
Aminoacyltransferases , CD8-Positive T-Lymphocytes , Chemokines , Neoplasms , Aminoacyltransferases/genetics , Aminoacyltransferases/metabolism , Animals , CD8-Positive T-Lymphocytes/pathology , Chemokines/metabolism , Immunotherapy , Leukemic Infiltration , Mice , Mice, Knockout , Monocytes , Neoplasms/immunologyABSTRACT
Human genetics and preclinical studies have identified key contributions of TREM2 to several neurodegenerative conditions, inspiring efforts to modulate TREM2 therapeutically. Here, we characterize the activities of three TREM2 agonist antibodies in multiple mixed-sex mouse models of Alzheimer's disease (AD) pathology and remyelination. Receptor activation and downstream signaling are explored in vitro, and active dose ranges are determined in vivo based on pharmacodynamic responses from microglia. For mice bearing amyloid-ß (Aß) pathology (PS2APP) or combined Aß and tau pathology (TauPS2APP), chronic TREM2 agonist antibody treatment had limited impact on microglia engagement with pathology, overall pathology burden, or downstream neuronal damage. For mice with demyelinating injuries triggered acutely with lysolecithin, TREM2 agonist antibodies unexpectedly disrupted injury resolution. Likewise, TREM2 agonist antibodies limited myelin recovery for mice experiencing chronic demyelination from cuprizone. We highlight the contributions of dose timing and frequency across models. These results introduce important considerations for future TREM2-targeting approaches.
Subject(s)
Alzheimer Disease , Membrane Glycoproteins , Microglia , Multiple Sclerosis , Receptors, Immunologic , Animals , Receptors, Immunologic/agonists , Receptors, Immunologic/metabolism , Receptors, Immunologic/genetics , Membrane Glycoproteins/agonists , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Mice , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Female , Male , Microglia/drug effects , Microglia/metabolism , Disease Models, Animal , Mice, Inbred C57BL , Mice, Transgenic , Antibodies/pharmacology , Humans , Amyloid beta-Peptides/metabolism , tau Proteins/metabolismABSTRACT
OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease1,2 and embryonic lethality during mouse development3. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice4 and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.
Subject(s)
Cell Death , Deubiquitinating Enzymes/metabolism , Endopeptidases/metabolism , Inflammation/metabolism , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitination , Animals , Caspase 8/genetics , Caspase 8/metabolism , Cell Death/genetics , Deubiquitinating Enzymes/genetics , Embryo Loss/genetics , Endopeptidases/genetics , Inflammation/enzymology , Inflammation/genetics , Interferon Type I/biosynthesis , Mice , Mice, Inbred C57BL , Receptor-Interacting Protein Serine-Threonine Kinases/deficiency , Receptor-Interacting Protein Serine-Threonine Kinases/genetics , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism , Ubiquitination/genetics , Weight Loss/geneticsABSTRACT
Parkinson's disease (PD), a neurodegenerative disease affecting dopaminergic (DA) neurons, is characterized by decline of motor function and cognition. Dopaminergic cell loss is associated with accumulation of toxic alpha synuclein aggregates. As DA neuron death occurs late in the disease, therapeutics that block the spread of alpha synuclein may offer functional benefit and delay disease progression. To test this hypothesis, we generated antibodies to the C terminal region of synuclein with high nanomolar affinity and characterized them in in vitro and in vivo models of spread. Interestingly, we found that only antibodies with high affinity to the distal most portion of the C-terminus robustly reduced uptake of alpha synuclein preformed fibrils (PFF) and accumulation of phospho (S129) alpha synuclein in cell culture. Additionally, the antibody treatment blocked the spread of phospho (S129) alpha synuclein associated-pathology in a mouse model of synucleinopathy. Blockade of neuronal PFF uptake by different antibodies was more predictive of in vivo activity than their binding potency to monomeric or oligomeric forms of alpha synuclein. These data demonstrate that antibodies directed to the C-terminus of the alpha synuclein have differential effects on target engagement and efficacy. Furthermore, our data provides additional support for the development of alpha synuclein antibodies as a therapeutic strategy for PD patients.
Subject(s)
Neurodegenerative Diseases , Parkinson Disease , Synucleinopathies , Mice , Animals , alpha-Synuclein/metabolism , Parkinson Disease/metabolism , Neurodegenerative Diseases/metabolism , Synucleinopathies/pathology , Dopaminergic Neurons/metabolismABSTRACT
The Abelson-helper integration site 1 (AHI1) gene encodes for a ciliary transition zone localizing protein that when mutated causes the human ciliopathy, Joubert syndrome. We prepared and examined neuronal cultures derived from male and female embryonic Ahi1+/+ and Ahi1-/- mice (littermates) and found that the distribution of ciliary melanin-concentrating hormone receptor-1 (MchR1) was significantly reduced in Ahi1-/- neurons; however, the total and surface expression of MchR1 on Ahi1-/- neurons was similar to controls (Ahi1+/+). This indicates that a pathway for MchR1 trafficking to the surface plasma membrane is intact, but the process of targeting MchR1 into cilia is impaired in Ahi1-deficient mouse neurons, indicating a role for Ahi1 in localizing MchR1 to the cilium. Mouse Ahi1-/- neurons that fail to accumulate MchR1 in the ciliary membrane have significant decreases in two downstream MchR1 signaling pathways [cAMP and extracellular signal-regulated kinase (Erk)] on MCH stimulation. These results suggest that the ciliary localization of MchR1 is necessary and critical for MchR1 signaling, with Ahi1 participating in regulating MchR1 localization to cilia, and further supporting cilia as critical signaling centers in neurons.SIGNIFICANCE STATEMENT Our work here demonstrates that neuronal primary cilia are powerful and focused signaling centers for the G-protein-coupled receptor (GPCR), melanin-concentrating hormone receptor-1 (MCHR1), with a role for the ciliary transition zone protein, Abelson-helper integration site 1 (AHI1), in mediating ciliary trafficking of MCHR1. Moreover, our manuscript further expands the repertoire of cilia functions on neurons, a cell type that has not received significant attention in the cilia field. Lastly, our work demonstrates the significant influence of ciliary GPCR signaling in the overall signaling of neurons.
Subject(s)
Adaptor Proteins, Vesicular Transport/physiology , Cilia/physiology , Neurons/physiology , Receptors, Somatostatin/physiology , Signal Transduction/physiology , Abnormalities, Multiple/genetics , Abnormalities, Multiple/physiopathology , Adaptor Proteins, Vesicular Transport/genetics , Animals , Cell Membrane/physiology , Cerebellum/abnormalities , Cerebellum/physiopathology , Cyclic AMP/metabolism , Eye Abnormalities/genetics , Eye Abnormalities/physiopathology , Female , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/physiopathology , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Mice , Mice, Knockout , Pregnancy , Receptors, Somatostatin/genetics , Retina/abnormalities , Retina/physiopathology , Signal Transduction/geneticsABSTRACT
Background and Objectives: Actinomyces species are part of the normal flora of humans and rarely cause disease. It is an uncommon cause of disease in humans. The clinical features of actinomycosis have been described, and various anatomical sites (such as face, bones and joints, respiratory tract, genitourinary tract, digestive tract, central nervous system, skin, and soft tissue structures) can be affected. It is not easy to identify actinomycosis because it sometimes mimics cancer due to under-recognition. As new diagnostic methods have been applied, Actinomyces can now more easily be identified at the species level. Recent studies have also highlighted differences among Actinomyces species. We report a case of Actinomyces viscosus bacteremia with cutaneous actinomycosis. Materials and Methods: A 66 years old male developed fever for a day with progressive right lower-leg erythematous swelling. Blood culture isolates yielded Actinomyces species, which was identified as Actinomyces viscosus by sequencing of the 16S rRNA gene. In addition, we searched for the term Actinomyces or actinomycosis cross-referenced with bacteremia or "blood culture" or "blood stream" from January 2010 to July 2020. The infectious diseases caused by species of A. viscosus from January 1977 to July 2020 were also reviewed. Results: The patient recovered well after intravenous ampicillin treatment. Poor oral hygiene was confirmed by dental examination. There were no disease relapses during the following period. Most cases of actinomycosis can be treated with penicillin. However, clinical alertness, risk factor evaluation, and identification of Actinomyces species can prevent inappropriate antibiotic or intervention. We also compiled a total of 18 cases of Actinomyces bacteremia after conducting an online database search. Conclusions: In summary, we describe a case of fever and progressive cellulitis. Actinomyces species was isolated from blood culture, which was further identified as Actinomyces viscosus by 16S rRNA sequencing. The cellulitis improved after pathogen-directed antibiotics. Evaluation of risk factors in patients with Actinomyces bacteremia and further identification of the Actinomyces species are recommended for successful treatment.
Subject(s)
Actinomycosis , Bacteremia , Actinomyces/genetics , Actinomyces viscosus , Actinomycosis/diagnosis , Actinomycosis/drug therapy , Aged , Bacteremia/diagnosis , Bacteremia/drug therapy , Humans , Male , RNA, Ribosomal, 16S/geneticsABSTRACT
BACKGROUND: Missense mutations in AHI1 result in the neurodevelopmental ciliopathy called Joubert syndrome. RESULTS: Mutations in AHI1 decrease cilia formation, alter its localization and stability, and change its binding to HAP1 and NPHP1. CONCLUSION: Mutations in AHI1 affect ciliogenesis, AHI1 protein localization, and AHI1-protein interactions. SIGNIFICANCE: This study begins to describe how missense mutations in AHI1 can cause Joubert syndrome. Mutations in AHI1 cause Joubert syndrome (JBTS), a neurodevelopmental ciliopathy, characterized by midbrain-hindbrain malformations and motor/cognitive deficits. Here, we show that primary cilia (PC) formation is decreased in fibroblasts from individuals with JBTS and AHI1 mutations. Most missense mutations in AHI1, causing JBTS, occur in known protein domains, however, a common V443D mutation in AHI1 is found in a region with no known protein motifs. We show that cells transfected with AHI1-V443D, or a new JBTS-causing mutation, AHI1-R351L, have aberrant localization of AHI1 at the basal bodies of PC and at cell-cell junctions, likely through decreased binding of mutant AHI1 to NPHP1 (another JBTS-causing protein). The AHI1-V443D mutation causes decreased AHI1 stability because there is a 50% reduction in AHI1-V443D protein levels compared with wild type AHI1. Huntingtin-associated protein-1 (Hap1) is a regulatory protein that binds Ahi1, and Hap1 knock-out mice have been reported to have JBTS-like phenotypes, suggesting a role for Hap1 in ciliogenesis. Fibroblasts and neurons with Hap1 deficiency form PC with normal growth factor-induced ciliary signaling, indicating that the Hap1 JBTS phenotype is likely not through effects at PC. These results also suggest that the binding of Ahi1 and Hap1 may not be critical for ciliary function. However, we show that HAP1 has decreased binding to AHI1-V443D indicating that this altered binding could be responsible for the JBTS-like phenotype through an unknown pathway. Thus, these JBTS-associated missense mutations alter their subcellular distribution and protein interactions, compromising functions of AHI1 in cell polarity and cilium-mediated signaling, thereby contributing to JBTS.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cerebellar Diseases/genetics , Eye Abnormalities/genetics , Kidney Diseases, Cystic/genetics , Mutation, Missense , Abnormalities, Multiple , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport , Amino Acid Sequence , Animals , Brain/metabolism , Brain/pathology , Cell Polarity , Cells, Cultured , Cerebellar Diseases/metabolism , Cerebellar Diseases/pathology , Cerebellum/abnormalities , Cilia/metabolism , Cilia/pathology , Conserved Sequence , Cytoskeletal Proteins , Eye Abnormalities/metabolism , Eye Abnormalities/pathology , Fibroblasts/metabolism , Fibroblasts/pathology , Humans , Intercellular Junctions/metabolism , Kidney Diseases, Cystic/metabolism , Kidney Diseases, Cystic/pathology , Membrane Proteins/metabolism , Mice , Mice, Transgenic , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Interaction Maps , Protein Stability , Protein Transport , Retina/abnormalities , Retina/metabolism , Retina/pathology , Signal Transduction , Two-Hybrid System TechniquesABSTRACT
Objective: Absolute lymphocyte count (ALC) has been implicated with short-term outcomes in a number of diseases, and we aimed to investigate the association between week-one ALC and long-term mortality in patients who were admitted to the medical intensive care units (ICUs). Methods: We enrolled patients who were admitted to the medical ICUs at the Taichung Veterans General Hospital, a referral centre located in central Taiwan, between 2015 and 2020 to conduct this retrospective cohort study. The outcome of interest was long-term all-cause mortality, and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated to determine the association. Furthermore, we employed propensity score-matching (PSM) and weighting techniques, consisting of inverse probability of treatment weighting (IPTW) and covariate balancing propensity score (CBPS), to confirm the association between ALC and mortality. Results: A total of 5722 critically ill patients were enrolled, and the one-year mortality was 44.8%. The non-survivor group had a lower ALC (1549, 1027-2388 vs 1948, 1373-2743 counts/µL, p<0.01) compared with those in the survivor group. Cox regression showed that low ALC was independently associated with mortality (adjHR 1.091, 95% CI 1.050-1.134). Propensity score-based analyses demonstrated the robust association, with adjHRs in the original, PSM, IPTW, and CBPS populations of 1.327 (95% CI 1.224-1.438), 1.301 (95% CI 1.188-1.424), 1.292 (95% CI 1.186-1.407), and 1.297 (95% CI 1.191-1.412), respectively. Sensitivity analyses further showed that the association between low ALC and mortality existed in a dose-response manner. Conclusion: We found that low ALC was associated with long-term mortality in critically ill patients; further studies are warranted to validate and translate these findings into clinical utility.
ABSTRACT
Eight gene clusters responsible for synthesizing bioactive metabolites associated with plant growth promotion were identified in the Bacillus cereus strain D1 (BcD1) genome using the de novo whole-genome assembly method. The two largest gene clusters were responsible for synthesizing volatile organic compounds (VOCs) and encoding extracellular serine proteases. The treatment with BcD1 resulted in an increase in leaf chlorophyll content, plant size, and fresh weight in Arabidopsis seedlings. The BcD1-treated seedlings also accumulated higher levels of lignin and secondary metabolites including glucosinolates, triterpenoids, flavonoids, and phenolic compounds. Antioxidant enzyme activity and DPPH radical scavenging activity were also found to be higher in the treated seedlings as compared with the control. Seedlings pretreated with BcD1 exhibited increased tolerance to heat stress and reduced disease incidence of bacterial soft rot. RNA-seq analysis showed that BcD1 treatment activated Arabidopsis genes for diverse metabolite synthesis, including lignin and glucosinolates, and pathogenesis-related proteins such as serine protease inhibitors and defensin/PDF family proteins. The genes responsible for synthesizing indole acetic acid (IAA), abscisic acid (ABA), and jasmonic acid (JA) were expressed at higher levels, along with WRKY transcription factors involved in stress regulation and MYB54 for secondary cell wall synthesis. This study found that BcD1, a rhizobacterium producing VOCs and serine proteases, is capable of triggering the synthesis of diverse secondary metabolites and antioxidant enzymes in plants as a defense strategy against heat stress and pathogen attack.
ABSTRACT
Molecular design and precise control of thin-film morphology and crystallinity of solution-processed small molecules are important for enhancing charge transport mobility of organic field-effect transistors and gaining more insight into the structure-property relationship. Here, two donor-acceptor-donor (D-A-D) architecture small molecules TRA-IID-TRA and TRA-TIID-TRA comprising an electron-donating triarylamine (TRA) and two different electron-withdrawing cores, isoindigo (IID) and thienoisoindigo (TIID), respectively, were synthesized and characterized. Replacing the phenylene rings of central IID A with thiophene gives a TIID core, which reduces the optical band gap and upshifts the energy levels of frontier molecular orbitals. The single-crystal structures and grazing-incidence wide-angle X-ray scattering (GIWAXS) analysis revealed that TRA-TIID-TRA exhibits the relatively tighter π-π stacking packing with preferential edge-on orientation, larger coherence length, and higher crystallinity due to the noncovalent S···O/S···π intermolecular interactions. The distinctly oriented and connected ribbon-like TRA-TIID-TRA crystalline film by the solution-shearing process achieved a superior hole mobility of 0.89 cm2 V-1 s-1 in the organic field-effect transistor (OFET) device, which is at least five times higher than that (0.17 cm2 V-1 s-1) of TRA-IID-TRA with clear cracks. Eventually, rational modulation of fused core in the π-conjugated D-A-D small molecule provides a new understanding of structural design for enhancing the performance of solution-processed organic semiconductors.
ABSTRACT
Purpose: Since there was no consensus on treatment options for localized prostate cancer, we performed a retrospective study to compare the long-term survival benefit of radiotherapy (RT) versus laparoscopic radical prostatectomy (LRP) in Taiwan. Methods: 218 patients with clinically localized prostate cancer treated between 2008 and 2017 (64 with LRP and 154 with RT) were enrolled in this study. The outcomes of RT and LRP were assessed after patients were stratified according to Gleason score, stage, and risk group. Crude survival, prostate cancer-specific survival, and metastasis-free survival were evaluated using the log-rank test. Results: The 5-year crude survival rate was 93.3% in the LRP group and 59.3% in the RT group. A significant survival benefit was found in the LRP group compared with the RT group (p = 0.004). Furthermore, significant differences were found in disease-specific survival (93.3% vs. 64.7%, p = 0.022) and metastasis-free survival (48% vs. 40.2%, p = 0.045) between the LRP and RT groups. Conclusions: Men with localized prostate cancer treated initially with LRP had a lower risk of prostate cancer-specific death and metastases compared with those treated with RT.
ABSTRACT
Vertebrate photoreceptors have a modified cilium composed of a basal body, axoneme and outer segment. The outer segment includes stacked membrane discs, containing opsin and the signal transduction apparatus mediating phototransduction. In photoreceptors, two distinct classes of vesicles are trafficked. Synaptic vesicles are transported down the axon to the synapse, whereas opsin-containing vesicles are transported to the outer segment. The continuous replacement of the outer segments imposes a significant biosynthetic and trafficking burden on the photoreceptors. Here, we show that Ahi1, a gene that when mutated results in the neurodevelopmental disorder, Joubert syndrome (JBTS), is required for photoreceptor sensory cilia formation and the development of photoreceptor outer segments. In mice with a targeted deletion of Ahi1, photoreceptors undergo early degeneration. Whereas synaptic proteins are correctly trafficked, photoreceptor outer segment proteins fail to be transported appropriately or are significantly reduced in their expression levels (i.e., transducin and Rom1) in Ahi1(-/-) mice. We show that vesicular targeting defects in Ahi1(-/-) mice are cilium specific, and our evidence suggests that the defects are caused by a decrease in expression of the small GTPase Rab8a, a protein required for accurate polarized vesicular trafficking. Thus, our results suggest that Ahi1 plays a role in stabilizing the outer segment proteins, transducin and Rom1, and that Ahi1 is an important component of Rab8a-mediated vesicular trafficking in photoreceptors. The retinal degeneration observed in Ahi1(-/-) mice recapitulates aspects of the retinal phenotype observed in patients with JBTS and suggests the importance of Ahi1 in photoreceptor function.
Subject(s)
Proto-Oncogene Proteins/metabolism , Retinal Degeneration/metabolism , Retinal Photoreceptor Cell Outer Segment/metabolism , Adaptor Proteins, Vesicular Transport , Animals , Brain Diseases , Cilia/metabolism , Eye Proteins/metabolism , GTP-Binding Protein alpha Subunits/metabolism , Gene Deletion , Membrane Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Knockout , Proto-Oncogene Proteins/genetics , Retina/metabolism , Synaptic Vesicles/metabolism , Syndrome , Tetraspanins , Transducin/metabolism , rab GTP-Binding Proteins/metabolismABSTRACT
The primary non-motile cilium, a membrane-ensheathed, microtubule-bundled organelle, extends from virtually all cells and is important for development. Normal functioning of the cilium requires proper axoneme assembly, membrane biogenesis and ciliary protein localization, in tight coordination with the intraflagellar transport system and vesicular trafficking. Disruptions at any level can induce severe alterations in cell function, giving rise to a myriad of human genetic diseases known as ciliopathies. Here we show that the Abelson helper integration site 1 (Ahi1) gene, whose human ortholog is mutated in Joubert syndrome, regulates cilium formation via its interaction with Rab8a, a small GTPase critical for polarized membrane trafficking. We find that the Ahi1 protein localizes to a single centriole, the mother centriole, which becomes the basal body of the primary cilium. In order to determine whether Ahi1 functions in ciliogenesis, loss of function analysis of Ahi1 was performed in cell culture models of ciliogenesis. Knockdown of Ahi1 expression by shRNAi in cells or targeted deletion of Ahi1 (Ahi1 knockout mouse) leads to impairments in ciliogenesis. In Ahi1-knockdown cells, Rab8a is destabilized and does not properly localize to the basal body. Since Rab8a is implicated in vesicular trafficking, we next examined this process in Ahi1-knockdown cells. Defects in the trafficking of endocytic vesicles from the plasma membrane to the Golgi and back to the plasma membrane were observed in Ahi1-knockdown cells. Overall, our data indicate that the distribution and functioning of Rab8a is regulated by Ahi1, not only affecting cilium formation, but also vesicle transport.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Cilia/metabolism , Mutation , Nervous System Diseases/metabolism , Proto-Oncogene Proteins/metabolism , Transport Vesicles/metabolism , rab GTP-Binding Proteins/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport , Animals , Cell Line , Cells, Cultured , Cilia/genetics , Female , Fibroblasts/metabolism , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nervous System Diseases/genetics , Protein Binding , Protein Transport , Proto-Oncogene Proteins/genetics , rab GTP-Binding Proteins/geneticsABSTRACT
Periventricular heterotopia (PH) is a disorder characterized by neuronal nodules, ectopically positioned along the lateral ventricles of the cerebral cortex. Mutations in either of two human genes, Filamin A (FLNA) or ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2), cause PH (Fox et al. in 'Mutations in filamin 1 prevent migration of cerebral cortical neurons in human periventricular heterotopia'. Neuron, 21, 1315-1325, 1998; Sheen et al. in 'Mutations in ARFGEF2 implicate vesicle trafficking in neural progenitor proliferation and migration in the human cerebral cortex'. Nat. Genet., 36, 69-76, 2004). Recent studies have shown that mutations in mitogen-activated protein kinase kinase kinase-4 (Mekk4), an indirect interactor with FlnA, also lead to periventricular nodule formation in mice (Sarkisian et al. in 'MEKK4 signaling regulates filamin expression and neuronal migration'. Neuron, 52, 789-801, 2006). Here we show that neurons in post-mortem human PH brains migrated appropriately into the cortex, that periventricular nodules were primarily composed of later-born neurons, and that the neuroependyma was disrupted in all PH cases. As studied in the mouse, loss of FlnA or Big2 function in neural precursors impaired neuronal migration from the germinal zone, disrupted cell adhesion and compromised neuroepithelial integrity. Finally, the hydrocephalus with hop gait (hyh) mouse, which harbors a mutation in Napa [encoding N-ethylmaleimide-sensitive factor attachment protein alpha (alpha-SNAP)], also develops a progressive denudation of the neuroepithelium, leading to periventricular nodule formation. Previous studies have shown that Arfgef2 and Napa direct vesicle trafficking and fusion, whereas FlnA associates dynamically with the Golgi membranes during budding and trafficking of transport vesicles. Our current findings suggest that PH formation arises from a final common pathway involving disruption of vesicle trafficking, leading to impaired cell adhesion and loss of neuroependymal integrity.
Subject(s)
Cerebral Ventricles/cytology , Periventricular Nodular Heterotopia/pathology , Stem Cells/cytology , Adult , Aged, 80 and over , Animals , Cell Adhesion , Cell Movement , Cerebral Ventricles/physiopathology , Contractile Proteins/genetics , Contractile Proteins/metabolism , Female , Filamins , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Humans , Infant, Newborn , Male , Mice , Mice, Transgenic , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , Neurons/physiology , Periventricular Nodular Heterotopia/physiopathology , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolismABSTRACT
Antibodies from B-cell clonal lineages share sequence and structural properties as well as epitope specificity. Clonally unrelated antibodies can similarly share sequence and specificity properties and are said to be convergent. Convergent antibody responses against several antigens have been described in humans and mice and include different classes of shared sequence features. In particular, some antigens and epitopes can induce convergent responses of clonally unrelated antibodies with restricted heavy (VH) and light (VL) chain variable region germline segment usage without similarity in the heavy chain third complementarity-determining region (CDR H3), a critical specificity determinant. Whether these V germline segment-restricted responses reflect a general epitope specificity restriction of antibodies with shared VH/VL pairing is not known. Here, we investigated this question by determining patterns of antigen binding competition between clonally unrelated antigen-specific rat antibodies from paired-chain deep sequencing datasets selected based solely on VH/VL pairing. We found that antibodies with shared VH/VL germline segment pairings but divergent CDR H3 sequences almost invariably have restricted epitope specificity indicated by shared binding competition patterns. This epitope restriction included 82 of 85 clonally unrelated antibodies with 13 different VH/VL pairings binding in 8 epitope groups in 2 antigens. The corollary that antibodies with shared VH/VL pairing and epitope-restricted binding can accommodate widely divergent CDR H3 sequences was confirmed by in vitro selection of variants of anti-human epidermal growth factor receptor 2 antibodies known to mediate critical antigen interactions through CDR H3. Our results show that restricted epitope specificity determined by VH/VL germline segment pairing is a general property of rodent antigen-specific antibodies.
Subject(s)
Antibody Specificity/immunology , Epitopes/immunology , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Amino Acid Sequence , Animals , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/immunology , RatsABSTRACT
Traditional hybridoma and B cell cloning antibody discovery platforms have inherent limits in immune repertoire sampling depth. One consequence is that monoclonal antibody (mAb) leads often lack the necessary affinity for therapeutic applications, thus requiring labor-intensive and time-consuming affinity in vitro engineering optimization steps. Here, we show that high-affinity variants of mouse-derived mAbs can be rapidly obtained by testing of somatic sequence variants obtained by deep sequencing of antibody variable regions in immune repertories from immunized mice, even with a relatively sparse sampling of sequence variants from large sequence datasets. Affinity improvements can be achieved for mAbs with a wide range of affinities. The optimized antibody variants derived from immune repertoire mining have no detectable in vitro off-target binding and have in vivo clearance comparable to the parental mAbs, essential properties in therapeutic antibody leads. As generation of antibody variants in vitro is replaced by mining of variants generated in vivo, the procedure can be applied to rapidly identify affinity-optimized mAb variants.
Subject(s)
Antibodies, Monoclonal/metabolism , B-Lymphocytes/immunology , Immunoglobulin Variable Region/genetics , Parkinson Disease/therapy , alpha-Synuclein/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Affinity , Clone Cells , High-Throughput Nucleotide Sequencing , Humans , Hybridomas , Immunization , Mice , Mice, Inbred C57BL , Parkinson Disease/immunology , Somatic Hypermutation, ImmunoglobulinABSTRACT
Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% (n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% (n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.
Subject(s)
Antibodies/metabolism , Antigens/metabolism , High-Throughput Nucleotide Sequencing/methods , Receptors, Antigen, B-Cell/genetics , Single-Cell Analysis , Animals , Germ Cells/metabolism , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Mice , Rats , Reproducibility of ResultsABSTRACT
Receptor-interacting protein kinase 4 (RIPK4) is a highly conserved regulator of epidermal differentiation. Members of the RIPK family possess a common kinase domain as well as unique accessory domains that likely dictate subcellular localization and substrate preferences. Mutations in human RIPK4 manifest as Bartsocas-Papas syndrome (BPS), a genetic disorder characterized by severe craniofacial and limb abnormalities. We describe the structure of the murine Ripk4 (MmRipk4) kinase domain, in ATP- and inhibitor-bound forms. The crystallographic dimer of MmRipk4 is similar to those of RIPK2 and BRAF, and we show that the intact dimeric entity is required for MmRipk4 catalytic activity through a series of engineered mutations and cell-based assays. We also assess the impact of BPS mutations on protein structure and activity to elucidate the molecular origins of the disease.
Subject(s)
Adenosine Triphosphate/metabolism , Mutation , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/metabolism , Animals , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation , Mice , Models, Molecular , Protein Conformation , Protein Multimerization , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins B-raf/chemistry , Receptor-Interacting Protein Serine-Threonine Kinase 2/chemistryABSTRACT
Polarized vesicle trafficking is mediated by small GTPase proteins, such as Rabs and Arls/Arfs. These proteins have essential roles in maintaining normal cellular function, in part, through regulating intracellular trafficking. Moreover, these families of proteins have recently been implicated in the formation and function of the primary cilium. The primary cilium, which is found on almost every cell type in vertebrates, is an organelle that protrudes from the surface of the cell and functions as a signaling center. Interestingly, it has recently been linked to a variety of human diseases, collectively referred to as ciliopathies. The primary cilium has an exceptionally high density of receptors on its membrane that are important for sensing and transducing extracellular stimuli. Moreover, the primary cilium serves as a separate cellular compartment from the cytosol, providing for unique spatial and temporal regulation of signaling molecules to initiate downstream events. Thus, functional primary cilia are essential for normal signal transduction. Rabs and Arls/Arfs play critical roles in early cilia formation but are also needed for maintenance of ciliary function through their coordination with intraflagellar transport (IFT), a specialized trafficking system in primary cilia. IFT in cilia is pivotal for the proper movement of proteins into and out of this highly regulated organelle. In this review article, we explore the involvement of polarized vesicular trafficking in cilia formation and function, and discuss how defects in these processes could subsequently lead to the abnormalities observed in ciliopathies.
ABSTRACT
Joubert syndrome (JBTS) is an autosomal recessive disorder characterized by cerebellum and brainstem malformations. Individuals with JBTS have abnormal breathing and eye movements, ataxia, hypotonia, and cognitive difficulty, and they display mirror movements. Mutations in the Abelson-helper integration site-1 gene (AHI1) cause JBTS in humans, suggesting that AHI1 is required for hindbrain development; however AHI1 may also be required for neuronal function. Support for this idea comes from studies demonstrating that the AHI1 locus is associated with schizophrenia. To gain further insight into the function of AHI1 in both the developing and mature central nervous system, we determined the spatial and temporal expression patterns of the gene products of AHI1 orthologs throughout development, in human, mouse, and zebrafish. Murine Ahi1 was distributed throughout the cytoplasm, dendrites, and axons of neurons, but was absent in glial cells. Ahi1 expression in the mouse brain was observed as early as embryonic day 10.5 and persisted into adulthood, with peak expression during the first postnatal week. Murine Ahi1 was observed in neurons of the hindbrain, midbrain, and ventral forebrain. Generally, the AHI1/Ahi1/ahi1 orthologs had a conserved distribution pattern in human, mouse, and zebrafish, but mouse Ahi1 was not present in the developing and mature cerebellum. Ahi1 was also observed consistently in the stigmoid body, a poorly characterized cytoplasmic organelle found in neurons. Overall, these results suggest roles for AHI1 in neurodevelopmental processes that underlie most of the neuroanatomical defects in JBTS, and perhaps in neuronal functions that contribute to schizophrenia.