Live-cell Immunofluorescence Research Articles

Inhibition of Autophagy Alters Intracellular Transport ofAPP Resulting in Increased APP Processing.

Alzheimer's disease (AD) pathology is characterized by amyloid beta (Aβ) plaques and dysfunctional autophagy. Aβ is generated by sequential proteolytic cleavage of amyloid precursor protein (APP), and the site of intracellular APP processing is highly debated, which may include autophagosomes. Here, we investigated the involvement of autophagy, including the role of ATG9 in APP intracellular trafficking and processing by applying the RUSH system, which allows studying the transport of fluorescently labeled mCherry-APP-EGFP in a systematic way, starting from the endoplasmic reticulum. HeLa cells, expressing the RUSH mCherry-APP-EGFP system, were investigated by live cell imaging, immunofluorescence, and Western blot. We found that mCherry-APP-EGFP passed through the Golgi faster in ATG9 knockout cells. Furthermore, ATG9 deletion shifted mCherry-APP-EGFP from early endosomes and lysosomes toward the plasma membrane concomitant with reduced endocytosis. Importantly, this alteration in mCherry-APP-EGFP transport resulted in increased secreted mCherry-soluble APP and C-terminal fragment-EGFP. These effects were also phenocopied by pharmacological inhibition of ULK1, indicating that autophagy is regulating the intracellular trafficking and processing of APP. These findings contribute to the understanding of the role of autophagy in APP metabolism and could potentially have implications for new therapeutic approaches for AD.

Caspase-mediated AURKA cleavage at Asp132 is essential for paclitaxel to elicit cell apoptosis.

Background: Aurora kinase A (AURKA) is a potent oncogene that is often aberrantly expressed during tumorigenesis, and is associated with chemo-resistance in various malignancies. However, the role of AURKA in chemo-resistance remains largely elusive. Methods: The cleavage of AURKA upon viral infection or apoptosis stimuli was assesed by immunoblotting assays in several cancer cells or caspase deficient cell line models. The effect of AURKA cleavage at Asp132 on mitosis was explored by live cell imaging and immunofluorescence staining experiments. The role of Asp132-cleavage of AURKA induced by the chemotherapy drug paclitaxel was investigated using TUNEL, immunohistochemistry assay in mouse tumor xenograft model and patient tissues. Results: The proteolytic cleavage of AURKA at Asp132 commonly occurs in several cancer cell types, regardless of viral infection or apoptosis stimuli. Mechanistically, caspase 3/7/8 cleave AURKA at Asp132, and the Asp132-cleaved forms of AURKA promote cell apoptosis by disrupting centrosome formation and bipolar spindle assembly in metaphase during mitosis. The AURKAD132A mutation blocks the expression of cleaved caspase 3 and EGR1, which leads to reduced therapeutic effects of paclitaxel on colony formation and malignant growth of tumor cells in vitro and in vivo using a murine xenograft model and cancer patients. Conclusions: This study reveals that caspase-mediated AURKAD132 proteolysis is essential for paclitaxel to elicit cell apoptosis and indicates that AURKAD132 is a potential key target for chemotherapy.

Discovery and Quantitative Dissection of Cytokinesis Mechanisms Using Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum is a versatile model for understanding many different cellular processes involving cell motility including chemotaxis, phagocytosis, and cytokinesis. Cytokinesis, in particular, is a model cell-shaped change process in which a cell separates into two daughter cells. D. discoideum has been used extensively to identify players in cytokinesis and understand how they comprise the mechanosensory and biochemical pathways of cytokinesis. In this chapter, we describe how we use cDNA library complementation with D. discoideum to discover potential regulators of cytokinesis. Once identified, these regulators are further analyzed through live cell imaging, immunofluorescence imaging, fluorescence correlation and cross-correlation spectroscopy, micropipette aspiration, and fluorescence recovery after photobleaching. Collectively, these methods aid in detailing the mechanisms and signaling pathways that comprise cell division.

RIT1 regulates mitosis and promotes proliferation by interacting with SMC3 and PDS5 in hepatocellular carcinoma

BackgroundAs a small G protein of Ras family, Ras-like-without-CAAX-1 (RIT1) plays a critical role in various tumors. Our previous study has demonstrated the involvement of RIT1 in promoting malignant progression of hepatocellular carcinoma (HCC). However, its underlying mechanism remains unclear.MethodsGene set enrichment analysis (GSEA) was conducted in the TCGA LIHC cohort to investigate the underlying biological mechanism of RIT1. Live cell imaging, immunofluorescence (IF) and flow cytometry assays were used to verify biological function of RIT1 in HCC mitosis. Subcutaneous xenografting of human HCC cells in BALB/c nude mice was utilized to assess tumor proliferation in vivo. RNA-seq, co-immunoprecipitation (Co-IP), mass spectrometry analyses, western blot and IF assays were employed to elucidate the mechanisms by which RIT1 regulates mitosis and promotes proliferation in HCC.ResultsOur findings demonstrate that RIT1 plays a crucial role in regulating mitosis in HCC. Knockdown of RIT1 disrupts cell division, leading to G2/M phase arrest, mitotic catastrophe, and apoptosis in HCC cells. SMC3 is found to interact with RIT1 and knockdown of SMC3 attenuates the proliferative effects mediated by RIT1 both in vitro and in vivo. Mechanistically, RIT1 protects and maintains SMC3 acetylation by binding to SMC3 and PDS5 during mitosis, thereby promoting rapid cell division and proliferation in HCC. Notably, we have observed an upregulation of SMC3 expression in HCC tissues, which is associated with poor patient survival and promotion of HCC cell proliferation. Furthermore, there is a significant positive correlation between the expression levels of RIT1, SMC3, and PDS5. Importantly, HCC patients with high expression of both RIT1 and SMC3 exhibit worse prognosis compared to those with high RIT1 but low SMC3 expression.ConclusionsOur findings underscore the crucial role of RIT1 in regulating mitosis in HCC and further demonstrate its potential as a promising therapeutic target for HCC treatment.

Preparation and identification of a single domain antibody specific for adenovirus vectors and its application to the immunoaffinity purification of adenoviruses

Adenovirus belongs to the family of Adenoviridae. As a vaccine carrier, it has high safety and stimulates the body to produce cellular immunity and humoral immunity. This study prepared an adenoviral vector-specific single-domain antibody for use in adenovirus identification and purification. We successfully constructed a single domain antibody phage display library with a capacity of 1.8 × 109 by immunizing and cloning the VHH gene from Bactrian camel. After the second round of biopanning, clones specific for adenovirus were screened using phage ELISA. Twenty-two positive clones were obtained, and two clones with the highest binding affinity from ELISA were selected and named sdAb 5 and sdAb 31 for further application. The recombinant single-domain antibody was solublely expressed in E. coli and specifically bound to adenoviruses rAd26, ChAd63 and HAd5 in ELISA and live cell immunofluorescence assays. We established an effective method for immunoaffinity purification of adenovirus by immobilizing the single domain antibody to Sepharose beads, and it may be used to selectively capture adenoviruses from cell culture medium. The preparation of the adenovirus-specific single-domain antibody lays a foundation for the one-step immunoaffinity purification and identification of adenoviruses.

Attenuated cell-cycle division protein 2 and elevated mitotic roles of polo-like kinase 1 characterize deficient myoblast fusion in peripheral arterial disease

IntroductionWe propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. MethodsDifferentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). ResultsIschemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. ConclusionDifferentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD.

PL3.6 Targeting GSK-3 activity promotes mitotic catastrophe via centrosome destabilisation and enhances the effect of radiotherapy in glioma models

Abstract BACKGROUND Targeting kinases as regulators of cellular processes that drive cancer progression is a promising approach to improve patient outcome in GBM management. The glycogen synthase kinase 3 (GSK-3) plays a role in cancer progression and is known for its pro-proliferative activity in gliomas. The anti-proliferative and cytotoxic effects of the GSK-3 inhibitor AZD2858 were assessed in relevant in vitro and in vivo glioma models to confirm GSK-3 as a suitable target for improved single agent or combination treatments. MATERIAL AND METHODS The immortalised cell line U251 and the patient derived cell lines GBM1 and GBM4 were used in in vitro studies including MTT, clonogenic survival, live cell imaging, immunofluorescence microscopy and flow cytometry to assess the cytotoxic and anti-proliferative effects of AZD2858. Observed anti-proliferative effects were investigated by microarray technology for the identification of target genes with known roles in cell proliferation. Clinical relevance of targeting GSK-3 with the inhibitor either for single agent or combination treatment strategies was determined by subcutaneous and orthotopic in vivo modelling. Whole mount mass spectroscopy was used to confirm drug penetration in orthotopic tumour models. RESULTS AZD2858 was cytotoxic at low micromolar concentrations and at sub-micromolar concentrations (0.01 - 1.0 μM) induced mitotic defects in all cell lines examined. Prolonged mitosis, centrosome disruption/duplication and cytokinetic failure leading to cell death featured prominently among the cell lines concomitant with an observed S-phase arrest. No cytotoxic or anti-proliferative effect was observed in normal human astrocytes. Analysis of the RNA microarray screen of AZD2858 treated glioma cells revealed the dysregulation of mitosis-associated genes including ASPM and PRC1, encoding proteins with known roles in cytokinesis. The anti-proliferative and cytotoxic effect of AZD2858 was also confirmed in both subcutaneous and orthotopic in vivo models. In addition, combination treatment with AZD2858 enhanced clinically relevant radiation doses leading to reduced tumour volume and improved survival in orthotopic in vivo models. CONCLUSION GSK-3 inhibition with the small molecule inhibitor AZD2858 led to cell death in glioma stem cells preventing normal centrosome function and promoting mitotic failure. Normal human astrocytes were not affected by treatment with the inhibitor at submicromolar concentrations. Drug penetration was observed alongside an enhanced effect of clinical radiotherapy doses in vivo. The reported aberrant centrosomal duplication may be a direct consequence of failed cytokinesis suggesting a role of GSK-3 in regulation of mitosis in glioma. GSK-3 is a promising target for combination treatment with radiation in GBM management and plays a role in mitosis-associated events in glioma biology.

Abstract 2923: DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy

Abstract A novel, clinical-stage DNA-PK inhibitor, M3814, potently and selectively blocks the non-homologous end-joining pathway for repair of DNA double strand breaks (DSB) and synergizes with ionizing radiation (IR) and DSB-inducing chemotherapy. We have previously shown that M3814 boosts ATM/p53 signaling and reinforces cell cycle checkpoint controls in irradiated p53 wild-type cancer cells, inducing irreversible premature senescence. However, in the absence of functional p53 this protective mechanism is disrupted, cancer cells progress through the cell cycle with unrepaired DSBs, leading to aberrant mitotic division and ultimately cell death. Here, we investigated the mechanistic consequences of DNA-PK inhibition in p53-dysfunctional cancer cells exposed to IR. We found that cell cycle progression with persistent DSB damage, promoted by M3814, causes severe chromosome aberrations, misalignment, and mis-segregation. This leads to the formation of micronuclei after each abnormal mitotic division. Live cell imaging and immunofluorescence analysis revealed high numbers of γH2AX foci, marking unrepaired DSBs inside the micronuclei, and lamin-B1-deficient micronuclei lamina. Cyclic GMP-AMP synthase (cGAS) was found to colocalize with micronuclei, suggesting that compromised laminar integrity exposes damaged DNA to the cytosol and activates the STING pathway. Gene expression analyses revealed activation of over 60 immune-related genes in cells exposed to IR+M3814, consistent with a STING-driven inflammatory response. Indeed, many cyto-/chemokines with immunomodulatory properties were detected in culture media from IR+M3814 treated cells. In addition, elevated expression of PD-L1 protein was found in irradiated p53-deficient cancer cells exposed to M3814. Together, these results provided a clear rationale for combination with PD-L1 inhibitory therapy. Murine p53-mutant cancer cells, MC38, were grown subcutaneously bilaterally in syngeneic mice. One of the tumors was exposed to 5 x 3.6 Gy daily IR fractions given locally and daily oral M3814 at previously established efficacious doses, followed by three applications (every 3-4 days) of the PD-L1 inhibitory antibody, avelumab. The other tumors were left unirradiated to monitor for abscopal effect. Study results demonstrated a superior benefit of adding M3814 to the IR/avelumab combination, including enhancement of the abscopal effect of IR/avelumab combination. Altogether, our results revealed that selective DNA-PK inhibition by M3814 provides a powerful new mechanism for induction of micronucleation and STING inflammatory signaling in p53-disfunctional cancer cells and offers a new approach to combination radio-immunotherapy of cancer. Citation Format: Michael Carr, Astrid Zimmermann, Yige Guo, Xiaohong Liu, Patrick Steiner, Susanne Hahn, Frank Zenke, Andree Blaukat, Lyubomir T. Vassilev. DNA-PK inhibitor, M3814, is a potent inducer of inflammatory micronucleation in irradiated p53-deficient cancer cells: Implications for combination radio-immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2923.

Metabolic Cross-talk Between Human Bronchial Epithelial Cells and Internalized Staphylococcus aureus as a Driver for Infection*

Staphylococcus aureus is infamous for causing recurrent infections of the human respiratory tract. This is a consequence of its ability to adapt to different niches, including the intracellular milieu of lung epithelial cells. To understand the dynamic interplay between epithelial cells and the intracellular pathogen, we dissected their interactions over 4 days by mass spectrometry. Additionally, we investigated the dynamics of infection through live cell imaging, immunofluorescence and electron microscopy. The results highlight a major role of often overlooked temporal changes in the bacterial and host metabolism, triggered by fierce competition over limited resources. Remarkably, replicating bacteria reside predominantly within membrane-enclosed compartments and induce apoptosis of the host within ∼24 h post infection. Surviving infected host cells carry a subpopulation of non-replicating bacteria in the cytoplasm that persists. Altogether, we conclude that, besides the production of virulence factors by bacteria, it is the way in which intracellular resources are used, and how host and intracellular bacteria subsequently adapt to each other that determines the ultimate outcome of the infectious process.

The serine protease testisin is present on the surface of capacitated stallion spermatozoa and interacts with key zona pellucida binding proteins.

Serine proteases are emerging as important players in the spermatozoon's acquisition of functional competence. This study aimed to characterize the serine protease testisin (PRSS21) in stallion spermatozoa, examining its surface expression, possible origins in the testis and epididymis, and changes in response to capacitation and acrosome reaction, as well as its capacity to form high molecular weight complexes and interact with other proteins. The role of serine proteases in spontaneous capacitation and acrosome reaction of stallion spermatozoa was established using the serine protease inhibitor, AEBSF. Testisin localization, before and after exposure of stallion spermatozoa to capacitating conditions and calcium ionophore, was examined using live cell immunofluorescence and flow cytometry. Immunohistochemistry of testicular and epididymal tissues was used to further dissect the origins of sperm testisin. Testisin's participation in high molecular weight protein complexes and identification of its interacting partner proteins were investigated using Blue Native PAGE, co-immunoprecipitation, and mass spectrometry, with interrogation of protein-protein interaction databases and gene ontology analysis of partner proteins used to further explore the potential roles of the testisin-containing complex in sperm function. Testisin surface expression increased significantly in capacitated spermatozoa (p<0.001), increased further following acrosome reaction (p<0.01), and was localized to the equatorial region of the sperm head. Testisin was also detected in luminal fluid within the caput and corpus regions of the epididymis, epididymal spermatozoa, and epididymal epithelial cells. Testisin formed several multiprotein complexes; co-immunoprecipitation revealed interactions of testisin with a multitude of zona pellucida-binding proteins, including ZPBP, ZAN, acrosin, several heat-shock proteins, and components of the TCP1 complex. Testisin appears to form part of the zona pellucida-binding complex in stallion spermatozoa and may be involved in the proteolytic cascade that prepares the sperm surface for interaction with the oocyte.

Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA− RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA+ versus ACPA− RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Effects of new beta-type Ti-40Nb implant materials, brain-derived neurotrophic factor, acetylcholine and nicotine on human mesenchymal stem cells of osteoporotic and non osteoporotic donors.

IntroductionTreatment of osteoporotic fractures is still challenging and an urgent need exists for new materials, better adapted to osteoporotic bone by adjusted Young’s modulus, appropriate surface modification and pharmaceuticals.Materials and methodsTitanium-40-niobium alloys, mechanically ground or additionally etched and titanium-6-aluminium-4-vanadium were analyzed in combination with brain-derived neurotrophic factor, acetylcholine and nicotine to determine their effects on human mesenchymal stem cells in vitro over 21 days using lactate dehydrogenase and alkaline phosphatase assays, live cell imaging and immunofluorescence microscopy.ResultsCell number of human mesenchymal stem cells of osteoporotic donors was increased after 14 d in presence of ground titanium-40-niobium or titanium-6-aluminium-4-vanadium, together with brain-derived neurotrophic factor. Cell number of human mesenchymal stem cells of non osteoporotic donors increased after 21 d in presence of titanium-6-aluminium-4-vanadium without pharmaceuticals. No significant increase was measured for ground or etched titanium-40-niobium after 21 d. Osteoblast differentiation of osteoporotic donors was significantly higher than in non osteoporotic donors after 21 d in presence of etched, ground titanium-40-niobium or titanium-6-aluminium-4-vanadium accompanied by all pharmaceuticals tested. In presence of all alloys tested brain-derived neurotrophic factor, acetylcholine and nicotine increased differentiation of cells of osteoporotic donors and accelerated it in non osteoporotic donors.ConclusionWe conclude that ground titanium-40-niobium and brain-derived neurotrophic factor might be most suitable for subsequent in vivo testing.

Identification of transcriptional targets of GSK3 involved in glioblastoma invasion

Glioblastoma multiforme remains one of the most difficult to treat cancers due to the highly infiltrative nature of cancer cells. Targeting the invasive properties of glioblastoma presents a novel treatment avenue. Recently, specific GSK-3 beta (GSK3b) inhibitors such as Bio-Indirubin (BIO), have been described that inhibit the migratory activity of glioma cells. In this study, the GSK-3b downstream targets were investigated. Genome-wide transcriptional profiling was used in order to identify GSK3b-driven target genes, and a transcriptional signature for glioblastoma invasion. U251 cells and patient-derived GBM1 cells were treated with the GSK3b inhibitor BIO. Microarray data obtained was mined for deregulated genes with known migratory roles. The up-regulation or down-regulation of candidate genes was confirmed by Western blotting and immunofluorescence. Knockdown of two candidate genes by RNAi, ARHGAP12 and ARHGAP29 was performed, and the effects on invasive properties and characteristics were assessed by 3D spheroid assays, live cell imaging and immunofluorescence, in order to investigate the effects of knockdowns on invasion. We have identified an invasion signature downstream of GSK3b, which includes activators and inactivators of the Rho family of small GTPases. Knockdown of the Rac1 inactivator ARHGAP12 increased migration, whereas knockdown of the RhoA inactivator ARHGAP29 decreased migration as assessed by live cell imaging and 3D spheroid invasion assays. This study, which for the first time investigates downstream targets of GSK-3b in glioblastoma, has identified an invasion signature which includes regulators of the Rho family of small GTPases. Identified genes will be potential candidates for genetic involvement in glioblastoma progression, and candidate diagnostic and prognostic biomarkers. Candidates will be assessed as potential therapeutic targets.

Clinical spectrum and IgG subclass analysis of anti-myelin oligodendrocyte glycoprotein antibody-associated syndromes: a multicenter study

Anti-myelin oligodendrocyte glycoprotein antibodies (MOG-Ab) recently emerged as a potential biomarker in patients with inflammatory demyelinating diseases of the central nervous system. We here compare the clinical and laboratory findings observed in a cohort of MOG-Ab seropositive and seronegative cases and describe IgG subclass analysis results. Consecutive serum samples referred to Verona University Neuropathology Laboratory for aquaporin-4 (AQP4)-Ab and/or MOG-Ab testing were analysed between March 2014 and May 2017. The presence of AQP4-Ab was determined using a cell-based assay. A live cell immunofluorescence assay was used for the detection of MOG-IgG and IgG subclass analysis. Among 454 analysed samples, 29 were excluded due to AQP4-Ab positivity or to the final demonstration of a disorder not compatible with MOG-Ab. We obtained clinical data in 154 out of 425 cases. Of these, 22 subjects resulted MOG-Ab positive. MOG-Ab positive patients were mainly characterised by the involvement of the optic nerve and/or spinal cord. Half of the cases presented relapses and the recovery was usually partial. Brain MRI was heterogeneous while short lesions were the prevalent observation on spinal cord MRI. MOG-Ab titre usually decreased in non-relapsing cases. In all MOG-IgG positive cases, we observed IgG1 antibodies, which were predominant in most subjects. IgG2 (5/22), IgG3 (9/22) and IgG4 (3/22) antibodies were also detectable. We confirm that MOG-Ab-related syndromes have distinct features in the spectrum of demyelinating conditions, and we describe the possible role of the different IgG subclasses in this condition.

Progesterone analogue protects stressed photoreceptors via bFGF-mediated calcium influx.

Retinitis pigmentosa (RP) is a degenerative retinal disease leading to photoreceptor cell loss. In 2011, our group identified the synthetic progesterone 'Norgestrel' as a potential treatment for RP. Subsequent research showed Norgestrel to work through progesterone receptor membrane component 1 (PGRMC1) activation and upregulation of neuroprotective basic fibroblast growth factor (bFGF). Using trophic factor deprivation of 661W photoreceptor-like cells, we aimed to further elucidate the mechanism leading to Norgestrel-induced neuroprotection. In the present manuscript, we show by flow cytometry and live-cell immunofluorescence that Norgestrel induces an increase in cytosolic calcium in both healthy and stressed 661Ws over 24h. Specific PGRMC1 inhibition by AG205 (1μm) showed this rise to be PGRMC1-dependent, primarily utilizing calcium from extracellular sources, for blockade of L-type calcium channels by verapamil (50μm) prevented a Norgestrel-induced calcium influx in stressed cells. Calcium influx was also shown to be bFGF-dependent, for siRNA knock down of bFGF prevented Norgestrel-PGRMC1 induced changes in cytosolic calcium. Notably, we demonstrate PGRMC1-activation is necessary for Norgestrel-induced bFGF upregulation. We propose that Norgestrel protects through the following pathway: binding to and activating PGRMC1 expressed on the surface of photoreceptor cells, PGRMC1 activation drives bFGF upregulation and subsequent calcium influx. Importantly, raised intracellular calcium is critical to Norgestrel's protective efficacy, for extracellular calcium chelation by EGTA abrogates the protective effects of Norgestrel on stressed 661W cells invitro.

Splice variants of the CaV1.3 L-type calcium channel regulate dendritic spine morphology.

Dendritic spines are the postsynaptic compartments of glutamatergic synapses in the brain. Their number and shape are subject to change in synaptic plasticity and neurological disorders including autism spectrum disorders and Parkinson’s disease. The L-type calcium channel CaV1.3 constitutes an important calcium entry pathway implicated in the regulation of spine morphology. Here we investigated the importance of full-length CaV1.3L and two C-terminally truncated splice variants (CaV1.342A and CaV1.343S) and their modulation by densin-180 and shank1b for the morphology of dendritic spines of cultured hippocampal neurons. Live-cell immunofluorescence and super-resolution microscopy of epitope-tagged CaV1.3L revealed its localization at the base-, neck-, and head-region of dendritic spines. Expression of the short splice variants or deletion of the C-terminal PDZ-binding motif in CaV1.3L induced aberrant dendritic spine elongation. Similar morphological alterations were induced by co-expression of densin-180 or shank1b with CaV1.3L and correlated with increased CaV1.3 currents and dendritic calcium signals in transfected neurons. Together, our findings suggest a key role of CaV1.3 in regulating dendritic spine structure. Under physiological conditions it may contribute to the structural plasticity of glutamatergic synapses. Conversely, altered regulation of CaV1.3 channels may provide an important mechanism in the development of postsynaptic aberrations associated with neurodegenerative disorders.

607. Organotypic Culture of Adult Porcine Retina as an In Vitro Screening Model for AAV-Mediated Gene Therapy in Ophthalmology

Introduction: Gene therapy studies in vivo using animal models provide critical information regarding vector specificity, therapeutic efficacy, and safety. However, long durations to observe transgene expression, high degree of variability between animals, and reduced control of experimental conditions often leads to large sample sizes and expense. Organotypic cultures of the retina can provide an efficient in vitro model with a high degree of throughput and control that can be used for pharmacological screening. These explants preserve complex intercellular processes and communications among the neural retinal layers with minimal manipulation of tissue architecture and molecular pathways, making it a useful model in target-tissue validation of adeno-associated virus (AAV) vector variants. Porcine retina has similar anatomical and physiologic features to the human retina and is therefore a suitable surrogate for preclinical testing in ophthalmology. Methods: We have developed a porcine explant culture system from retinas isolated from adult pigs as a model to screen novel rAAV vectors for transduction efficiency and cellular tropism. Pig eyes were enucleated and the neural retina dissected away from the pigment epithelium within 1-2 hours postmortem. Tissue was cut into 4 mm pieces and placed photoreceptor layer down on polycarbonate transwells, and cultured in Neurobasal-A medium supplemented with B-27. Viability of the explant cultures was examined over time by gross morphology, quantification of glucose-6-phosphate dehydrogenase release from dying cells, and TUNEL assay. Transductions with AAV vectors were performed on retinal explants on the day the cultures were started, and transgene expression was analyzed by live cell imaging and immunofluorescence (IF) staining, or ELISA from tissue supernatants. Results: Retinal explants were viable for up to 4 weeks and IF staining of retinal protein markers including β-III-tubulin, Chx10, glutamine synthetase, rhodopsin and L/M opsin suggested that most retinal cell types were preserved in vitro, with tissue architecture closely resembling that of intact in vivo retina. Transduction with different rAAV serotypes encoding GFP displayed protein expression at 3 days, with gradually rising levels during the first 2 weeks following transduction. Furthermore, IF staining using retinal cell markers revealed differential cellular transgene expression from the AAV vectors encoding various cell-specific or ubiquitous promoters under investigation. Finally, high levels of secreted soluble VEGFR-1 protein expressed from rAAV were detected from explant culture media samples with expression increasing over a period of 3 weeks. Conclusions: Our results demonstrate viability of retinal explants for up to 4 weeks and efficient rAAV transduction with various serotypes. Our organotypic porcine retinal explant model facilitates the evaluation of efficacy, cellular tropism, and promoter selectivity of rAAV vectors in a fast, reproducible, and economical manner, contributing to a reduction in the use of animals for the development of therapies in ophthalmology.

MP70-05 DEVELOPMENT OF A NOVEL 3 DIMENSIONAL (3D) TESTICULAR ORGANOID MODEL FOR THE STUDY OF HUMAN SPERMATOGENESIS AND GENOTOXICITY IN VITRO

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology1 Apr 2016MP70-05 DEVELOPMENT OF A NOVEL 3 DIMENSIONAL (3D) TESTICULAR ORGANOID MODEL FOR THE STUDY OF HUMAN SPERMATOGENESIS AND GENOTOXICITY IN VITRO Samuel Pendergraft, Hooman Sadri-Ardekani, Tanya Reid, Anthony Atala, and Colin Bishop Samuel PendergraftSamuel Pendergraft More articles by this author , Hooman Sadri-ArdekaniHooman Sadri-Ardekani More articles by this author , Tanya ReidTanya Reid More articles by this author , Anthony AtalaAnthony Atala More articles by this author , and Colin BishopColin Bishop More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.1430AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Mammalian spermatogenesis is regulated through paracrine-endocrine activity, cell signaling, and local control mechanisms. These highly specific signaling interactions are effectively absent upon placing testicular cells into two-dimensional (2D) primary culture. Current protocols to produce mature germ cells in vitro are inefficient and are limited in supporting post-meiotic cells. In order to address these limitations we have developed a 3D testis organoid in vitro by combining stem cell and tissue engineering approaches. This model can be utilized as a means to evaluate gonadotoxic agents, and to address critical deficiencies in our understanding of basic human spermatogenesis Human testes tissues for this study were obtained through the National Disease Research Interchange (NDRI). METHODS Development of this model system consisted of (1) Identification and analysis of specific cellular components necessary for use in a 3D culture method, (2) establishment of basic design parameters, culture conditions, and characterization of human testicular organoids using live cell imaging, immunofluorescence, immunohistochemistry, gene expression, and viability assays, and (3) assess the potential to demonstrate testicular organoid function and as a tool for screening for testicular drug toxicity. RESULTS Human Spermatogonial stem cells (SSCs), Sertoli, and Leydig cells were isolated from human adult testes tissues, characterized, and expanded. These cell types were integrated successfully into 3D organoids and maintained viability as determined by ATP and Live/Dead assays for over 3 weeks in culture. These organoids were responsive to hCG and able to produce testosterone in vitro. Changes in gene expression within these multicellular human testis organoids was measured over time for cell and stage-specific markers including UCHL1, ITGA6, DAZL, SYCP3, PRM1, ACROSIN, FSHr, CYP19a1, HSD3B1, and CYP11a1. Dose response curves were generated using a luminescent viability assay in response to exposure to 3 commonly used genotoxic chemotherapeutic drugs and dose-response was compared to corresponding cells cultured in 2D. Calculated IC50 values were determined to be significantly higher for 3D organoid cultures. CONCLUSIONS Functional human 3D testicular organoids were successfully generated. Future directions include optimizing the spermatogenic capacity of the organoids and continuing to evaluate their use as a novel testicular toxicity model © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e907 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Samuel Pendergraft More articles by this author Hooman Sadri-Ardekani More articles by this author Tanya Reid More articles by this author Anthony Atala More articles by this author Colin Bishop More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

Antibodies to aquaporin-1 are not present in neuromyelitis optica.

Although more than 70% of all patients with neuromyelitis optica (NMO) are seropositive for aquaporin-4 (AQP4) antibodies, a substantial proportion of patients fulfilling the clinical criteria for NMO or limited forms of the disease (NMO spectrum disorders [NMOSD]) are negative for these antibodies.1 This raises the possibility of other autoantibodies in these patients. One putative target, the ubiquitously expressed water channel aquaporin-1 (AQP1), which is partially lost or internalized in certain NMO lesions,2 was described in a subset of patients with NMOSD.3–5 However, AQP1 antibodies were also found in patients with multiple sclerosis (MS), thus raising concerns about the specificity of these findings. Therefore, we developed a recombinant live cell immunofluorescence assay (CBA) for AQP1 antibodies based on our AQP4 antibody assay.6 We analyzed 176 serum samples from Austrian patients with NMOSD (n = 67), Austrian patients with MS (n = 31), and controls (n = 78) for the presence of IgG AQP1 antibodies and AQP4 antibodies. Furthermore, both assays were validated in a blinded cohort of 58 patients with NMOSD (n = 36) or MS (n = 22) from Lyon (France) and Oxford (United Kingdom). Clinical and demographic data of patients and controls are shown in the table. Acknowledgment: The authors are grateful to the Austrian NMO study group for contributing to this work.