Association of sedation and anesthesia on cognitive outcomes in very premature infants: a retrospective observational study.

PURPOSE: Cognitive outcomes in preterm infants may be adversely affected by use of sedation and anesthetic agents. We investigated the associations between anesthetics/sedatives and full-scale intelligence quotient (FSIQ) measured at 36 months corrected age (CA) in very preterm infants (born < 29 weeks gestational age). METHODS: This retrospective cohort study included preterm infants born at < 29 weeks of gestation between 1 January 2006 and 31 December 2012, whose cognitive outcomes were assessed at 36 months CA. Imputed and complete case univariable and adjusted multivariable linear regressions were used to investigate the associations between FSIQ [standardized to mean (standard deviation) 100 (15)] and exposure to volatile anesthetics, propofol, benzodiazepines, barbiturates, and ketamine. These agents were the subject of a 2016 warning from regulatory authorities in the USA recommending caution for administration to children and pregnant women. RESULTS: A total of 731 infants met the inclusion criteria. Unadjusted associations were -7 (95% confidence interval [CI], -10 to -4; P < 0.001) and -6 (95% CI, -10 to -3; P < 0.001) FSIQ points with exposure to warned medications using imputed and complete case analyses, respectively. Imputed and complete case adjusted associations between FSIQ and warned medications were -3 (95% CI, -7 to 0; P = 0.045) and -4 (95% CI, -8 to 0; P = 0.071) FSIQ points, respectively. Adjusted associations between volatile anesthetic exposure only and FSIQ were -3 (95% CI, -6 to 0; P = 0.072) and -5 (95% CI, -9 to -2; P = 0.004) FSIQ points using imputed and complete case data sets, respectively. FSIQ was not associated with opioid exposure. CONCLUSION: Exposure of very preterm infants to anesthetics/sedatives on the United States Food and Drug Administration warning list was associated with a decrease in FSIQ points at 36 months CA. There was no association between opioid exposure and FSIQ.

RéSUMé: OBJECTIF : L'utilization d'agents sédatifs et anesthésiques pourrait avoir une incidence défavorable sur l'évolution cognitive des nourrissons prématurés. Nous avons analysé les associations existantes entre les anesthésiques/sédatifs et le quotient d'intelligence global (QIg) mesuré à 36 mois d'âge corrigé (AC) chez des enfants nés grands prématurés (nés < 29 semaines d'âge gestationnel). MéTHODES: Cette étude de cohorte rétrospective a inclus des nourrissons prématurés nés avant 29 semaines d'âge gestationnel entre le 1er janvier 2006 et le 31 décembre 2012 et dont les critères d'évaluation cognitifs ont été évalués à 36 mois d'AC. Des régressions linéaires à une seule variable et multivariables ajustée, sur les cas imputés et sur les cas complets, ont été utilisées pour rechercher les associations entre le QIg (standardisé à la moyenne 100 [± écart-type] [15]) et l'exposition à des anesthésiques volatils, du propofol, des benzodiazépines, des barbituriques et de la kétamine. Ces molécules ont fait l'objet d'une mise en garde en 2016 par les autorités de réglementation aux États-Unis, recommandant la prudence concernant leur administration à des enfants et à des femmes enceintes. RéSULTATS: Un total de 731 nourrissons présentait les critères d'inclusion. Les associations non ajustées ont été de -7 (intervalle de confiance [IC] à 95 % : -10 à -4; P < 0,001) et -6 (IC à 95 % : -10 à -3; P < 0,001) points de QIg avec l'exposition aux médicaments sous avertissement en utilisant, respectivement, des analyses de cas imputés et de cas complets. Les associations ajustées de cas imputés et complets entre le QIg et les médicaments sous avertissement ont été, respectivement, de -3 (IC à 95 % : -7 à 0; P = 0,045) et -4 (IC à 95 % : -8 à 0; P = 0,071) points de QIg. Les associations ajustées entre l'exposition aux anesthésiques volatiles, uniquement, et le QIg ont été de -3 (IC à 95 % : -6 à 0; P = 0,072) et -5 (IC à 95 % : -9 à 2; P = 0,004) points de QIg en utilisant, respectivement, les ensembles de données des cas imputés et des cas complets. Le QIg n'a pas été associé à une exposition aux opioïdes. CONCLUSION: L'exposition des nourrissons grands prématurés aux anesthésiques/sédatifs figurant sur la liste d'avertissement de la Food and Drug Administration des États-Unis a été associée à une diminution des points de QIg à 36 mois d'AC. Il n'y a pas eu d'association entre l'exposition aux opioïdes et le QIg.

Case Report: Post obstructive pulmonary edema (POPE) Type II following elective adenotonsillectomy requiring novel use of high frequency oscillatory ventilation (HFOV).

This case report describes a previously healthy 14 year-old patient undergoing elective outpatient adenotonsillectomy that was complicated by acute postoperative pulmonary edema requiring 12 hours of high frequency oscillatory ventilation (HFOV) support.  We describe the clinical findings that led us to this rare diagnosis and management of post obstructive pulmonary edema (POPE) Type II, a rare but recognized complication following the surgical relief of an upper airway obstruction. This case is unique in that no previously published case report or review of POPE Type II has described the need for HFOV support.

Anesthetic Neuroprotection in Experimental Stroke in Rodents: A Systematic Review and Meta-analysis.

BACKGROUND: Patients undergoing endovascular therapy for acute ischemic stroke may require general anesthesia to undergo the procedure. At present, there is little clinical evidence to guide the choice of anesthetic in this acute setting. The clinical implications of experimental studies demonstrating anesthetic neuroprotection are poorly understood. Here, the authors evaluated the impact of anesthetic treatment on neurologic outcome in experimental stroke. METHODS: Controlled studies of anesthetics in stroke using the filament occlusion model were identified in electronic databases up to December 15, 2015. The primary outcome measures, infarct volume, and neurologic deficit score were used to calculate the normalized mean difference for each comparison. Meta-analysis of normalized mean difference values provided estimates of neuroprotection and contributions of predefined factors: study quality, the timing of treatment, and the duration of ischemia. RESULTS: In 80 retrieved publications anesthetic treatment reduced neurologic injury by 28% (95% CI, 24 to 32%; P < 0.0001). Internal validity was high: publication bias enhanced the effect size by 4% or less, effect size increased with study quality (P = 0.0004), and approximately 70% of studies were adequately powered. Apart from study quality, no predefined factor influenced neuroprotection. Neuroprotection failed in animals with comorbidities. Neuroprotection by anesthetics was associated with prosurvival mechanisms. CONCLUSIONS: Anesthetic neuroprotection is a robust finding in studies using the filament occlusion model of ischemic stroke and should be assumed to influence outcomes in studies using this model. Neuroprotection failed in female animals and animals with comorbidities, suggesting that the results in young male animals may not reflect human stroke.

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

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.

BACKGROUND: Primary 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. METHODS: Seven 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. RESULTS: We 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. CONCLUSIONS: Primary 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.

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

BACKGROUND: 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). METHODS: Studies were identified by searching the electronic databases OVID MEDLINE and Pubmed and scanning reference lists of articles by two authors. Limits were applied to the English language and to humans. We used the following search terms: CF, hemophilia A, ESRD, ESLD in combination with academic performance, educational status, educational measurement, learning, achievement, developmental delay, learning disabilities, intellectual disabilities, behavioral disorders, intelligence quotient (IQ), cognition, school problems, absenteeism, school attendance, anxiety, learning regression, or developmental regression. The search strategy was reviewed independently by all four authors. Eligibility assessment was performed independently in an unblinded standardized manner by two authors who chose relevant articles from the overall search results by scanning the titles and abstracts of articles and from the references within citations. The full-text publications were reviewed by all four authors. All pertinent data related to the objectives were collected and independently reviewed by two authors. The data were summarized in the form reported in the studies. When possible, reported data were submitted to analysis with the Mantel-Haenszel method using a random effects model. Analyses were performed using the Review Manager computer program. RESULTS: In the studies retrieved, the main outcomes were measures of intellectual or cognitive characteristics, as exemplified by the Wechsler battery of tests. Reporting of measures of achievement (for example, GPA) was rare. Children with CF and hemophilia A did not appear disadvantaged by their disease as general intelligence levels were comparable with the general population norms. In children with ESRD, mean IQ reported during dialysis improved after transplantation. Although they improved relative to their pretransplantation cognitive functioning, children with ESLD who received transplants are approximately eight IQ points below the population norm. CONCLUSIONS: Overall, the results suggest that the burden of chronic childhood illness, by itself, does not impair cognitive development in children with hemophilia A and CF. Children with ESRD and ESLD, despite optimal management, show a mild cognitive deficit compared with the population norm. Given the impact of these four specific chronic illnesses on neurocognitive outcome in children and the improvement in IQ post-transplant in both ESRD and ESLD, the results suggest that the effect of an uncontrolled confounding illness on neurocognitive development is small.

Relationship of other cytoplasmic ribonucleoprotein bodies (cRNPB) to GW/P bodies.

GW/P body components are involved in the post-transcriptional -processing of messenger RNA (mRNA) through the RNA interference and 5' → 3' mRNA degradation pathways, as well as functioning in mRNA transport and stabilization. It is currently thought that the relevant mRNA silencing and degrading factors are partitioned to these cytoplasmic microdomains thus effecting post-transcriptional regulation and the prevention of accidental degradation of functional mRNA. Although much attention has focused on GW/P bodies, a variety of other cytoplasmic RNP bodies (cRNPB) also have highly specialized functions and have been shown to interact or co-localize with components of GW/P bodies. These cRNPB include neuronal transport RNP granules, stress granules, RNP-rich cytoplasmic germline granules or chromatoid bodies, sponge bodies, cytoplasmic prion protein-induced RNP granules, U bodies and TAM bodies. Of clinical relevance, autoantibodies directed against protein and miRNA components of GW/P bodies have been associated with autoimmune diseases, neurological diseases and cancer. Understanding the molecular function of GW/P bodies and their interactions with other cRNPB may provide clues to the etiology or pathogenesis of diseases associated with autoantibodies directed to these structures. This chapter will focus on the similarities and differences of the various cRNPB as an approach to understanding their functional relationships to GW/P bodies.

An SNP in the trinucleotide repeat region of the TNRC6A gene maps to a major TNGW1 autoepitope in patients with autoantibodies to GW182.

GW/P bodies contain two TNRC6A protein isoforms (GW182 and TNGW1) that function as translational repressors of mRNA through Ago2-mediated RNA silencing. Autoantibodies to GW/P body components GW182, Ge-1 and Ago2 have previously been correlated with clinical autoimmune diseases including neurological disease, Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis and primary biliary cirrhosis. No studies were published to date examining if patients with autoantibodies directed against GW/P bodies contain autoantibodies to the trinucleotide repeat (TNR) region of TNGW1, which differs from GW182 only by the addition of an N-terminal QP-rich 253 amino acid sequence. Our data show that 85.7% of GW/P body positive plasma contain autoantibodies to various epitopes in the TNR region of TNGW1. Given the association of neurological diseases with autoantibodies directed to the TNR region on exon 5 of TNRC6A, this study examined whether there were TNR expansions as described in other neurological diseases and/or mutations in the nucleotide sequence of the CAG/CCA/G-rich region in seven anti-GW/P body positive patients, six control and eight breast cancer patients. Although a TNR expansion was not identified, 28.6% of patients containing autoantibodies to the TNR of TNGW1 were shown to have a single nucleotide polymorphism (SNP) at c.344C > A in the CAG/CCA/G-rich region of TNRC6A, which when translated, would produce a protein variant of p.Pro115Gln. The amino acid change may alter the structure of TNGW1 and/or perturb its miRNA regulatory function although this has not been examined experimentally. A putative change in protein structure may lead to a loss of tolerance to the TNGW1 protein or result in a "neo-antigen" in patients containing the specific TNRC6A SNPs. Further studies of a larger cohort of GW/P body positive patients and structure-function relationships of the variant TNRC6A are required to fully understand the role that such SNPs play in GW/P body autoantibody production and/or pathogenesis of related autoimmune diseases.

Repression of GW/P body components and the RNAi microprocessor impacts primary ciliogenesis in human astrocytes.

BACKGROUND: In most cells, the centriolar component of the centrosome can function as a basal body supporting the formation of a primary cilium, a non-motile sensory organelle that monitors information from the extracellular matrix and relays stimuli into the cell via associated signaling pathways. Defects in the formation and function of primary cilia underlie multiple human diseases and are hallmarks of malignancy. The RNA silencing pathway is involved in the post-transcriptional silencing of > 50% of mRNA that occurs within GW/P bodies. GW/P bodies are found throughout the cytoplasm and previously published live cell imaging data suggested that in a malignant cell type (U2OS), two GW/P bodies reside at the centrosome during interphase. This led us to investigate if a similar relationship exists in primary cells and if the inhibition of the miRNA pathway impairs primary cilium formation. RESULTS: Two GW/P bodies as marked by GW182 and hAgo2 colocalized to the basal body of primary human astrocytes as well as human synoviocytes during interphase and specifically with the distal end of the basal body in the pericentriolar region. Since it is technically challenging to examine the two centrosomal GW/P bodies in isolation, we investigated the potential relationship between the global population of GW/P bodies and primary ciliogenesis. Astrocytes were transfected with siRNA directed to GW182 and hAgo2 and unlike control astrocytes, a primary cilium was no longer associated with the centrosome as detected in indirect immunofluorescence assays. Ultrastructural analysis of siRNA transfected astrocytes revealed that knock down of GW182, hAgo2, Drosha and DGCR8 mRNA did not affect the appearance of the earliest stage of ciliogenesis but did prevent the formation and elongation of the ciliary axoneme. CONCLUSIONS: This study confirms and extends a previously published report that GW/P bodies reside at the centrosome in U2OS cells and documents that GW/P bodies are resident at the centrosome in diverse non-malignant cells. Further, our study demonstrates that repression of key effector proteins in the post-transcriptional miRNA pathway impairs primary cilium formation.

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.

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.

Cytoplasmic ribonucleoprotein (RNP) bodies and their relationship to GW/P bodies.

GW bodies (glycine- and tryptophan-rich cytoplasmic bodies; also known as mammalian processing (P) or Dcp-containing bodies) were described in 2002 when a human autoimmune serum was used to immunoscreen a HeLa expression library. Subsequently, many investigators have focused their attention on elucidating the components and functional relevance of this ribonucleoprotein (RNP)-containing cytoplasmic microdomain to cellular and molecular biology, developmental and pathological processes, and clinical practice. GW/P body components are now known to be involved in the post-transcriptional processing of messenger RNA (mRNA) through the RNA interference pathway, 5'-->3' mRNA degradation as well as mRNA transport and stabilization. It is currently thought that the relevant mRNA silencing and degrading factors are partitioned to these restricted cytoplasmic microdomains thus effecting post-transcriptional regulation and the prevention of accidental degradation of functional mRNA. Although much attention has focused on GW/P bodies, other cytoplasmic RNP bodies, which have highly specialized functions, interact or co-localize with components of GW/P bodies. These include neuronal transport RNP granules, stress granules, RNP-rich cytoplasmic germline granules or chromatoid bodies, sponge bodies, cytoplasmic prion protein-induced RNP granules, U bodies and TAM bodies. This review will focus on the similarities and differences of the various cytoplasmic RNP granules as an approach to understanding their functional relationships to GW/P bodies.

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

BACKGROUND: Primary 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. METHODS: Cultured 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. RESULTS: We 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. CONCLUSIONS: The 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.

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.

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.

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 approximately 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.