Joanna J. Moser

Adenylate cyclase regulates elongation of mammalian primary cilia

The primary cilium is a non-motile microtubule-based structure that shares many similarities with the structures of flagella and motile cilia. It is well known that the length of flagella is under stringent control, but it is not known whether this is true for primary cilia. In this study, we found that the length of primary cilia in fibroblast-like synoviocytes, either in log phase culture or in quiescent state, was confined within a range. However, when lithium was added to the culture to a final concentration of 100 mM, primary cilia of synoviocytes grew beyond this range, elongating to a length that was on average approximately 3 times the length of untreated cilia. Lithium is a drug approved for treating bipolar disorder. We dissected the molecular targets of this drug, and observed that inhibition of adenylate cyclase III (ACIII) by specific inhibitors mimicked the effects of lithium on primary cilium elongation. Inhibition of GSK-3beta by four different inhibitors did not induce primary cilia elongation. ACIII was found in primary cilia of a variety of cell types, and lithium treatment of these cell types led to their cilium elongation. Further, we demonstrate that different cell types displayed distinct sensitivities to the lithium treatment. However, in all cases examined primary cilia elongated as a result of lithium treatment. In particular, two neuronal cell types, rat PC-12 adrenal medulla cells and human astrocytes, developed long primary cilia when lithium was used at or close to the therapeutic relevant concentration (1-2 mM). These results suggest that the length of primary cilia is controlled, at least in part, by the ACIII-cAMP signaling pathway.

Primary ciliogenesis defects are associated with human astrocytoma/glioblastoma cells.

BackgroundPrimary cilia are non-motile sensory cytoplasmic organelles that have been implicated in signal transduction, cell to cell communication, left and right pattern embryonic development, sensation of fluid flow, regulation of calcium levels, mechanosensation, growth factor signaling and cell cycle progression. Defects in the formation and/or function of these structures underlie a variety of human diseases such as Alström, Bardet-Biedl, Joubert, Meckel-Gruber and oral-facial-digital type 1 syndromes. The expression and function of primary cilia in cancer cells has now become a focus of attention but has not been studied in astrocytomas/glioblastomas. To begin to address this issue, we compared the structure and expression of primary cilia in a normal human astrocyte cell line with five human astrocytoma/glioblastoma cell lines.MethodsCultured normal human astrocytes and five human astrocytoma/glioblastoma cell lines were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy. Monospecific antibodies were used to detect primary cilia and map the relationship between the primary cilia region and sites of endocytosis.ResultsWe show that expression of primary cilia in normal astrocytes is cell cycle related and the primary cilium extends through the cell within a unique structure which we show to be a site of endocytosis. Importantly, we document that in each of the five astrocytoma/glioblastoma cell lines fully formed primary cilia are either expressed at a very low level, are completely absent or have aberrant forms, due to incomplete ciliogenesis.ConclusionsThe recent discovery of the importance of primary cilia in a variety of cell functions raises the possibility that this structure may have a role in a variety of cancers. Our finding that the formation of the primary cilium is disrupted in cells derived from astrocytoma/glioblastoma tumors provides the first evidence that altered primary cilium expression and function may be part of some malignant phenotypes. Further, we provide the first evidence that ciliogenesis is not an all or none process; rather defects can arrest this process at various points, particularly at the stage subsequent to basal body association with the plasma membrane.

Identification of GW182 and its novel isoform TNGW1 as translational repressors in Ago2-mediated silencing.

RNA interference is triggered by small interfering RNA and microRNA, and is a potent mechanism in post-transcriptional regulation for gene expression. GW182 (also known as TNRC6A), an 182-kDa protein encoded by TNRC6A, is important for this process, although details of its function remain unclear. Here, we report a novel 210-kDa isoform of human GW182, provisionally named trinucleotide GW1 (TNGW1) because it contains trinucleotide repeats in its mRNA sequence. TNGW1 was expressed independently of GW182 and was present in human testis and various human cancer cells. Using polyclonal and monoclonal antibodies, we detected TNGW1 in only ∼30% of GW bodies. Expression of EGFP-tagged TNGW1 in HeLa cells was colocalized to cytoplasmic foci enriched in Ago2 (also known as EIF2C2) and RNA decay factors. Tethering TNGW1 or GW182 to the 3′-UTR of a luciferase-reporter mRNA led to strong repression activity independent of Ago2, whereas the tethered Ago2-mediated suppression was completely dependent on TNGW1 and/or GW182. Our data demonstrated that GW182 and, probably, TNGW1 acted as a repressor in Ago2-mediated translational silencing. Furthermore, TNGW1 might contribute to diversity in the formation and function of GW and/or P bodies.

Markers of mRNA stabilization and degradation, and RNAi within astrocytoma GW bodies.

GW bodies (GWBs) are unique cytoplasmic structures that contain the mRNA binding protein GW182 and other proteins involved in mRNA processing pathways. The rationale for this study arose from clinical studies indicating that 33% of patients with GWB autoantibodies have a motor/sensory neuropathy and/or ataxia. The novelty of this study is the identification of GWBs in astrocytes and astrocytoma cells within cell bodies and cytoplasmic projections. Astrocytoma GWBs exhibit complex heterogeneity with combinations of LSm4 and XRN1 as well as Ago2 and Dicer, key proteins involved in mRNA degradation and RNA interference, respectively. GWB subsets contained the mRNA transport and stabilization proteins SYNCRIP, hnRNPA1, and FMRP, not previously described as part of the GWB complex. Immunoprecipitation of astrocytoma GWBs suggested that Dicer, hDcp, LSm4, XRN1, SYNCRIP, and FMRP form a multiprotein complex. GWBs are likely involved in a number of regulatory mRNA pathways in astrocytes and astrocytoma cells.

The PCM–basal body/primary cilium coalition

The centrosome is an organelle that acts as a microtubule-organizing center (MTOC) throughout the cell cycle. Within the centrosome are often two components that each have an ability to organize microtubule structures: the centriole that has the potential to function as a basal body and nucleate a cilium or a flagellum and a mass of protein material that in the presence of a centriole is commonly referred to as the pericentriolar material (PCM) that organizes cytoplasmic and spindle microtubule arrays. One characteristic of a large variety of cells is the ability to express a non-motile primary cilium. It is now appreciated that the function of the primary cilium is integral to a variety of essential cell functions and defects affecting this structure underlie a variety of human disease. While the function of the primary cilium and manner in which a basal body organizes a primary cilium has received extensive attention there is now a need to explore the inter-relationship between the PCM and the basal body/primary cilium. It is this latter topic that is the focus of this review where we show that the PCM is integrated with the centriole to form a coalition that is essential for both the expression and function of the primary cilium as well as the organization and function of the cellular environment that surrounds it.

A systematic review and quantitative analysis of neurocognitive outcomes in children with four chronic illnesses

Concern has been expressed that infants and children exposed to uneventful surgery and anesthesia may incur neurological injury that becomes manifest in poor scholastic performance or future learning difficulties. A recent meta‐analysis of seven clinical studies examined the relationship between learning or behavior difficulties and pediatric exposure to anesthesia/surgery and reported an odds ratio of 1.4; however, the level of association and causal factors remain unclear. The purpose of our study is to provide context to the pediatric anesthesia neurotoxicity question by reviewing the evidence linking four childhood illnesses with neurocognitive development. In the present review, we have sought to quantify the magnitude of the impact of chronic illness on neurocognitive development through a systematic review of publications that report the developmental trajectory of patients with four childhood diseases: cystic fibrosis (CF), hemophilia A, end‐stage renal disease (ESRD) and end‐stage liver disease (ESLD).

The MicroRNA and MessengerRNA Profile of the RNA-Induced Silencing Complex in Human Primary Astrocyte and Astrocytoma Cells

Background GW/P bodies are cytoplasmic ribonucleoprotein-rich foci involved in microRNA (miRNA)-mediated messenger RNA (mRNA) silencing and degradation. The mRNA regulatory functions within GW/P bodies are mediated by GW182 and its binding partner hAgo2 that bind miRNA in the RNA-induced silencing complex (RISC). To date there are no published reports of the profile of miRNA and mRNA targeted to the RISC or a comparison of the RISC-specific miRNA/mRNA profile differences in malignant and non-malignant cells. Methodology/Principal Findings RISC mRNA and miRNA components were profiled by microarray analysis of malignant human U-87 astrocytoma cells and its non-malignant counterpart, primary human astrocytes. Total cell RNA as well as RNA from immunoprecipitated RISC was analyzed. The novel findings were fourfold: (1) miRNAs were highly enriched in astrocyte RISC compared to U-87 astrocytoma RISC, (2) astrocytoma and primary astrocyte cells each contained unique RISC miRNA profiles as compared to their respective cellular miRNA profiles, (3) miR-195, 10b, 29b, 19b, 34a and 455-3p levels were increased and the miR-181b level was decreased in U-87 astrocytoma RISC as compared to astrocyte RISC, and (4) the RISC contained decreased levels of mRNAs in primary astrocyte and U-87 astrocytoma cells. Conclusions/Significance The observation that miR-34a and miR-195 levels were increased in the RISC of U-87 astrocytoma cells suggests an oncogenic role for these miRNAs. Differential regulation of mRNAs by specific miRNAs is evidenced by the observation that three miR34a-targeted mRNAs and two miR-195-targeted mRNAs were downregulated while one miR-195-targeted mRNA was upregulated. Biological pathway analysis of RISC mRNA components suggests that the RISC plays a pivotal role in malignancy and other conditions. This study points to the importance of the RISC and ultimately GW/P body composition and function in miRNA and mRNA deregulation in astrocytoma cells and possibly in other malignancies.

Optimization of immunoprecipitation–western blot analysis in detecting GW182-associated components of GW/P bodies

Characterizing the components of GW/processing bodies is key to elucidating RNA interference and messenger RNA processing pathways. This protocol addresses challenges in isolating a low-abundance protein GW182 and GW body (GWB)-associated proteins by building on previous reports that used polyclonal sera containing autoantibodies to GW/P body components. This protocol uses commercially available monoclonal antibodies to GW182 that are covalently coupled to Protein A or G sepharose beads and then used to immunoprecipitate GW182 and associated proteins from cell extracts. Immunoprecipitates are separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and probed by western blot with antibodies directed to proteins of interest. This protocol, which is expected to take 4–5 d, provides a biochemical approach for detecting GW182 and associated proteins in biological samples and thus facilitates the elucidation of the diverse functions of GWBs. It is expected that this protocol can be adapted to the detection of other RNA-binding complexes.

Evidence for Epigenetic Regulation of Gene Expression and Function in Chronic Experimental Diabetic Neuropathy.

Abstract Diabetic polyneuropathy (DPN) is a common but irreversible neurodegenerative complication of diabetes mellitus. Here we show that features of sensory neuron damage in mice with chronic DPN may have altered epigenetic micro RNA (miRNA) transcriptional control. We profiled sensory neuron messenger RNA and miRNA profiles in mice with type I diabetes mellitus and findings of DPN. Diabetic sensory dorsal root ganglia neurons showed a pattern of altered messenger RNA profiles associated with upregulated cytoplasmic sites of miRNA-mediated messenger RNA processing (GW/P bodies). Dorsal root ganglia miRNA microarray identified significant changes in expression among mice with diabetes, the most prominent of which were a 39% downregulation of mmu-let-7i and a 255% increase in mmu-miR-341; both were identified in sensory neurons. To counteract these alterations, we replenished let-7i miRNA by intranasal administration; in a separate experiment, we added an anti-miR that antagonized elevated mmu-341 after 5 months of diabetes. Both approaches independently improved electrophysiologic, structural, and behavioral abnormalities without altering hyperglycemia; control sequences did not have these effects. Dissociated adult sensory neurons exposed to an exogenous mmu-let-7i mimic displayed enhanced growth and branching, indicating a trophic action. These findings identify roles for epigenetic miRNA alterations and enhanced GW/P expression in diabetic dorsal root ganglia that contribute to the complex DPN phenotype.

Ultrastructural characterization of primary cilia in pathologically characterized human glioblastoma multiforme (GBM) tumors

BackgroundPrimary cilia are non-motile sensory cytoplasmic organelles that are involved in cell cycle progression. Ultrastructurally, the primary cilium region is complex, with normal ciliogenesis progressing through five distinct morphological stages in human astrocytes. Defects in early stages of ciliogenesis are key features of astrocytoma/glioblastoma cell lines and provided the impetus for the current study which describes the morphology of primary cilia in molecularly characterized human glioblastoma multiforme (GBM) tumors.MethodsSeven surgically resected human GBM tissue samples were molecularly characterized according to IDH1/2 mutation status, EGFR amplification status and MGMT promoter methylation status and were examined for primary cilia expression and structure using indirect immunofluorescence and electron microscopy.ResultsWe report for the first time that primary cilia are disrupted in the early stages of ciliogenesis in human GBM tumors. We confirm that immature primary cilia and basal bodies/centrioles have aberrant ciliogenesis characteristics including absent paired vesicles, misshaped/swollen vesicular hats, abnormal configuration of distal appendages, and discontinuity of centriole microtubular blades. Additionally, the transition zone plate is able to form in the absence of paired vesicles on the distal end of the basal body and when a cilium progresses beyond the early stages of ciliogenesis, it has electron dense material clumped along the transition zone and a darkening of the microtubules at the proximal end of the cilium.ConclusionsPrimary cilia play a role in a variety of human cancers. Previously primary cilia structure was perturbed in cultured cell lines derived from astrocytomas/glioblastomas; however there was always some question as to whether these findings were a cell culture phenomena. In this study we confirm that disruptions in ciliogenesis at early stages do occur in GBM tumors and that these ultrastructural findings bear resemblance to those previously observed in cell cultures. This is the first study to demonstrate that defects in cilia expression and function are a true hallmark of GBM tumors and correlate with their unrestrained growth. A review of the current ultrastructural profiles in the literature provides suggestions as to the best possible candidate protein that underlies defects in the early stages of ciliogenesis within GBM tumors.