Publications by authors named "Stephanie Carvalho"

24 Publications

  • Page 1 of 1

Multi-cohort profiling reveals elevated CSF levels of brain-enriched proteins in Alzheimer's disease.

Ann Clin Transl Neurol 2021 Jul 15;8(7):1456-1470. Epub 2021 Jun 15.

Division of Affinity Proteomics, Department of Protein Science, SciLifeLab, KTH Royal Institute of Technology, Stockholm, Sweden.

Objective: Decreased amyloid beta (Aβ) 42 together with increased tau and phospho-tau in cerebrospinal fluid (CSF) is indicative of Alzheimer's disease (AD). However, the molecular pathophysiology underlying the slowly progressive cognitive decline observed in AD is not fully understood and it is not known what other CSF biomarkers may be altered in early disease stages.

Methods: We utilized an antibody-based suspension bead array to analyze levels of 216 proteins in CSF from AD patients, patients with mild cognitive impairment (MCI), and controls from two independent cohorts collected within the AETIONOMY consortium. Two additional cohorts from Sweden were used for biological verification.

Results: Six proteins, amphiphysin (AMPH), aquaporin 4 (AQP4), cAMP-regulated phosphoprotein 21 (ARPP21), growth-associated protein 43 (GAP43), neurofilament medium polypeptide (NEFM), and synuclein beta (SNCB) were found at increased levels in CSF from AD patients compared with controls. Next, we used CSF levels of Aβ42 and tau for the stratification of the MCI patients and observed increased levels of AMPH, AQP4, ARPP21, GAP43, and SNCB in the MCI subgroups with abnormal tau levels compared with controls. Further characterization revealed strong to moderate correlations between these five proteins and tau concentrations.

Interpretation: In conclusion, we report six extensively replicated candidate biomarkers with the potential to reflect disease development. Continued evaluation of these proteins will determine to what extent they can aid in the discrimination of MCI patients with and without an underlying AD etiology, and if they have the potential to contribute to a better understanding of the AD continuum.
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http://dx.doi.org/10.1002/acn3.51402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8283172PMC
July 2021

Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues.

Methods Mol Biol 2021 ;2276:67-85

Federal University of Rio de Janeiro, Institute of Medical Biochemistry Leopoldo de Meis, Rio De Janeiro, RJ, Brazil.

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.
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http://dx.doi.org/10.1007/978-1-0716-1266-8_5DOI Listing
July 2021

Comprehensive Quantitative Proteome Analysis of Identifies Proteins and Pathways Involved in and Zika Virus Interference Phenomenon.

Front Physiol 2021 25;12:642237. Epub 2021 Feb 25.

Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

Zika virus (ZIKV) is a global public health emergency due to its association with microcephaly, Guillain-Barré syndrome, neuropathy, and myelitis in children and adults. A total of 87 countries have had evidence of autochthonous mosquito-borne transmission of ZIKV, distributed across four continents, and no antivirus therapy or vaccines are available. Therefore, several strategies have been developed to target the main mosquito vector, , to reduce the burden of different arboviruses. Among such strategies, the use of the maternally-inherited endosymbiont has been applied successfully to reduce virus susceptibility and decrease transmission. However, the mechanisms by which orchestrate resistance to ZIKV infection remain to be elucidated. In this study, we apply isobaric labeling quantitative mass spectrometry (MS)-based proteomics to quantify proteins and identify pathways altered during ZIKV infection; infection; co-infection with ZIKV in the heads and salivary glands. We show that regulates proteins involved in reactive oxygen species production, regulates humoral immune response, and antioxidant production. The reduction of ZIKV polyprotein in the presence of in mosquitoes was determined by MS and corroborates the idea that helps to block ZIKV infections in The present study offers a rich resource of data that may help to elucidate mechanisms by which orchestrate resistance to ZIKV infection in , and represents a step further on the development of new targeted methods to detect and quantify ZIKV and directly in complex tissues.
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http://dx.doi.org/10.3389/fphys.2021.642237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7947915PMC
February 2021

Aedes aegypti post-emergence transcriptome: Unveiling the molecular basis for the hematophagic and gonotrophic capacitation.

PLoS Negl Trop Dis 2021 01 6;15(1):e0008915. Epub 2021 Jan 6.

Instituto de Bioquímica Médica Leopoldo de Meis, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Rio de Janeiro, Brazil.

The adult females of Aedes aegypti mosquitoes are facultative hematophagous insects but they are unable to feed on blood right after pupae emergence. The maturation process that takes place during the first post-emergence days, hereafter named hematophagic and gonotrophic capacitation, comprises a set of molecular and physiological changes that prepare the females for the first gonotrophic cycle. Notwithstanding, the molecular bases underlying mosquito hematophagic and gonotrophic capacitation remain obscure. Here, we investigated the molecular and biochemical changes in adult Ae. aegypti along the first four days post-emergence, prior to a blood meal. We performed a RNA-Seq analysis of the head and body, comparing male and female gene expression time courses. A total of 811 and 203 genes were differentially expressed, respectively in the body and head, and both body parts showed early, mid, and late female-specific expression profiles. Female-specific up-regulation of genes involved in muscle development and the oxidative phosphorylation pathway were remarkable features observed in the head. Functional assessment of mitochondrial oxygen consumption in heads showed a gradual increase in respiratory capacity and ATP-linked respiration as a consequence of induced mitochondrial biogenesis and content over time. This pattern strongly suggests that boosting oxidative phosphorylation in heads is a required step towards blood sucking habit. Several salivary gland genes, proteases, and genes involved in DNA replication and repair, ribosome biogenesis, and juvenile hormone signaling were up-regulated specifically in the female body, which may reflect the gonotrophic capacitation. This comprehensive description of molecular and biochemical mechanisms of the hematophagic and gonotrophic capacitation in mosquitoes unravels potentially new targets for vector control.
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http://dx.doi.org/10.1371/journal.pntd.0008915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815146PMC
January 2021

Cerebrospinal Fluid Levels of Kininogen-1 Indicate Early Cognitive Impairment in Parkinson's Disease.

Mov Disord 2020 11 15;35(11):2101-2106. Epub 2020 Aug 15.

Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.

Background: Cognitive impairment is common in patients with PD. Core markers of Alzheimer's dementia have been related also to PD dementia, but no disease-specific signature to predict PD dementia exists to date.

Objectives: The aim of this study was to investigate CSF markers associated with cognition in early PD.

Methods: A high-throughput suspension bead array examined 216 proteins in CSF of 74 PD patients in the AETIONOMY project. Cognitive function was assessed with Repeatable Battery for the Assessment of the Neuropsychological Status, Montreal Cognitive Assessment, and Mini-Mental State Examination.

Results: Of 69 patients with complete data, 34 had high (≥90) and 35 had low Repeatable Battery for the Assessment of the Neuropsychological Status total score (<90). Of 14 proteins in CSF that differed in levels between groups, increased kininogen-1, validated with several antibodies, was independently associated with lower Repeatable Battery for the Assessment of the Neuropsychological Status and Montreal Cognitive Assessment scores after adjustment for confounders.

Conclusions: Kininogen-1 levels in CSF may serve as a marker of cognitive impairment in PD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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http://dx.doi.org/10.1002/mds.28192DOI Listing
November 2020

Description of Referrals for Colposcopy in a Hospital in Brazil.

Rev Bras Ginecol Obstet 2020 Mar 31;42(3):140-145. Epub 2020 Mar 31.

Department of Gynecology, Universidade de Taubaté, Taubaté, SP, Brazil.

Objective:  To describe the referral for colposcopy in a Hospital in Brazil and the relative frequency of patients who benefited from it, considering the correct indications for the examination and its final diagnoses.

Methods:  A retrospective study was performed in the colposcopy service database of the Hospital Universitário de Taubaté, Taubaté, state of São Paulo, Brazil. The frequency validated in the analysis of the medical records of women referred for clinical indication or cytological alteration, attended from March 2015 to March 2017. The population selected and analyzed included 256 results that were correlated to the cytological, clinical data and the result of the colposcopy.

Results:  Of the women referred, 45% presented out of the age of screening according to the guidelines of cervical cancer screening, 8.6% being adolescents and young adults < 25 years old, and 36.4% of the patients being ≥ 65 years old. A total of 50% of the patients had no indication of colposcopy, that is, normal cytologies, benign changes, ectopia, cervicitis, atypical squamous cells of indeterminate significance (ASC-US) and low-grade intraepithelial lesion (LSIL) without persistence and normal clinical appearance. A total of 39.84% who underwent colposcopy had high-grade lesion or cancer results, thus benefiting from the adequate referral.

Conclusion:  Most (60.16%) of the patients referred to the colposcopy service did not benefit from the referral for results without changes, such as negative colposcopies, histologies with no cervical intraepithelial neoplasm (CIN) or only CIN 1, or were out of the age for screening. These findings therefore demonstrate a significant number of unnecessary and inadequate referrals.
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http://dx.doi.org/10.1055/s-0040-1708886DOI Listing
March 2020

Multicenter Alzheimer's and Parkinson's disease immune biomarker verification study.

Alzheimers Dement 2020 02 6;16(2):292-304. Epub 2020 Jan 6.

Department of Neurodegenerative Diseases & Geropsychiatry/Neurology, University of Bonn Medical Center, Bonn, Germany.

Introduction: Multiple immunity biomarkers have been suggested as tracers of neuroinflammation in neurodegeneration. This study aimed to verify findings in cerebrospinal fluid (CSF) samples of Alzheimer's disease (AD) and Parkinson's disease (PD) subjects from the network of the European, Innovative Medicines Initiative-funded project AETIONOMY.

Methods: A total of 227 samples from the studies/centres AETIONOMY, ICEBERG, and IDIBAPS were used to analyse 21 selected immunity biomarkers in CSF. Results were compared to data of an independent cohort of 399 subjects previously published.

Results: Immunity markers were predominantly and reproducibly associated with pathological levels of tau isoforms, but also with amyloid levels, aging, sex, APOE genotype, and center-specific factors.

Discussion: Immunity biomarker levels in CSF reflect molecular and cellular pathology rather than diagnosis in neurodegenerative disorders. Assay standardization and stratification for age and other covariates could improve the power of such markers in clinical applications or intervention studies targeting immune responses in neurodegeneration.
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http://dx.doi.org/10.1016/j.jalz.2019.07.018DOI Listing
February 2020

Use of extracorporeal bypass is associated with improved outcomes in open thoracic and thoracoabdominal aortic aneurysm repair.

J Vasc Surg 2018 10 31;68(4):941-947. Epub 2018 Mar 31.

Division of Vascular Surgery and Endovascular Interventions, Columbia University Medical Center, Columbia University College of Physicians and Surgeons, New York, NY. Electronic address:

Objective: There is no consensus on the use or benefit of extracorporeal circulation (EC) during aneurysm repair of the descending thoracic aorta (DTA) or thoracoabdominal aorta (TAA). We evaluated the role of EC during DTA or TAA aneurysm repair using U.S. Medicare data.

Methods: Medicare (2004-2007) patients undergoing open repair of nonruptured DTA or TAA aneurysm were identified by International Classification of Diseases, Ninth Revision code. Specific exclusions included ascending aortic or arch repairs, concomitant cardiac procedures, and procedures employing deep hypothermic circulatory arrest. The impact of EC (code 3961) on early and late outcomes was analyzed using univariate analysis and multivariable regression. Survival was assessed using Kaplan-Meier analysis and Cox proportional hazards regression models.

Results: There were 4230 patients who had repair of intact DTA or TAA aneurysms, 2433 (57%) of which employed EC. Differences in baseline clinical features of EC and non-EC patients showed that patients undergoing aortic reconstruction with EC were older (73 ± 1 years vs 72 ± 1 years; P = .002), were more likely to be female (53% vs 47%; P < .001), and had more hypertension (56% vs 53%; P = .02); they had less chronic obstructive pulmonary disease (28% vs 34%; P < .0001), peripheral vascular disease (5.7% vs 11.3%; P < .001), and chronic kidney disease (7.7% vs 5.5%; P = .003). The 30-day mortality (9.7% for EC vs 12.2%; P = .02) and any major complication (49% for EC vs 58%; P < .001) were significantly reduced with EC use. EC use was associated with a shorter length of stay (13.5 ± 13 days vs 17.2 ± 18 days; P < .01) and lower total hospital charges ($151,000 ± 140,000 vs $180,000 ± 190,000; P < .01) compared with non-EC patients. EC patients were more likely to be discharged home instead of to an extended care facility (67% vs 56%; P < .01). Multivariable regression modeling to adjust for baseline clinical differences showed EC to independently reduce the risk of operative mortality (odds ratio [OR], 0.80; 95% confidence interval [CI], 0.65-0.97; P = .02), any complication (OR, 0.67; 95% CI, 0.59-0.76; P < .01), pulmonary complications (OR, 0.68; 95% CI, 0.59-0.79; P < .01), and acute renal failure (OR, 0.52; 95% CI, 0.44-0.61; P < .01). Long-term survival was higher (log-rank, P < .01) in EC patients at 1 year (81% ± 0.8% vs 73% ± 1%) and 5 years (67% ± 1% vs 52% ± 1%). Risk-adjusted Cox proportional hazards regression also showed that EC was independently associated with improved long-term survival (hazard ratio, 0.69; 95% CI, 0.63-0.74; P < .01).

Conclusions: Although important clinical variables such as DTA or TAA aneurysm extent and spinal cord ischemic complications cannot be assessed with the Medicare database, EC use during open DTA and TAA aneurysm repair is associated with improved late survival and a significant reduction in operative mortality, morbidity, and procedural costs. These data indicate that EC should be a more widely applied adjunct in open DTA or TAA aneurysm repair.
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http://dx.doi.org/10.1016/j.jvs.2017.12.072DOI Listing
October 2018

A Systematic Review of Integrated Care Interventions Addressing Perinatal Depression Care in Ambulatory Obstetric Care Settings.

Clin Obstet Gynecol 2018 09;61(3):573-590

Departments of Obstetrics and Gynecology.

This systematic review searched 4 databases (PubMed/MEDLINE, Scopus, CINAHL, and PsychINFO) and identified 21 articles eligible to evaluate the extent to which interventions that integrate depression care into outpatient obstetric practice are feasible, effective, acceptable, and sustainable. Despite limitations among the available studies including marked heterogeneity, there is evidence supporting feasibility, effectiveness, and acceptability. In general, this is an emerging field with promise that requires additional research. Critical to its real-world success will be consideration for practice workflow and logistics, and sustainability through novel reimbursement mechanisms.
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http://dx.doi.org/10.1097/GRF.0000000000000360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6059986PMC
September 2018

Factors related to non-adherence to mammography in a city of the Brazilian Amazonian area: A population-based study.

Rev Assoc Med Bras (1992) 2017 Jan;63(1):35-42

Graduate Program in Health Sciences, UFRR, Boa Vista, RR, Brazil.

Objective: To assess the prevalence of mammography use and factors related to non-adherence in Boa Vista, capital of Roraima, Brazil.

Method: A cross sectional study, quantitative analysis, based on household survey was performed between June and August 2013, using a face-to-face interview with a pre-tested form. Target population was women between 40 and 69 years. The sample size target was 240 participants, and the sampling method was random cluster sampling. The study was approved by the Institutional Review Board of Federal University of Roraima.

Results: 241 women were included without refusals. The prevalence of non-use of mammography in the past two years was 55.6% (95CI 49.1-61.9). In univariate analysis, the risk factors for non-adherence to mammography were having low educational level, family income below three minimum wages, receiving government assistance, not having consulted with a doctor and no health insurance. In multivariate analysis, only low educational level and receiving government assistance remained as risk factors. Medical consultation or health worker visiting were protective factors.

Conclusion: Adherence to mammography is unsatisfactory in Boa Vista, Roraima, and has a predominantly opportunistic character. Low educational level is confirmed as an independent risk factor, but belonging to a family that receives government assistance can be interpreted as a social marker of families and/or areas lacking of government intervention to increase access to breast cancer control programs.
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http://dx.doi.org/10.1590/1806-9282.63.01.35DOI Listing
January 2017

Multicenter clinical trial of the conformable stent graft for the treatment of acute, complicated type B dissection.

J Vasc Surg 2015 Aug;62(2):271-8

Division of Vascular Surgery, University of Wisconsin, Madison, Wisc.

Objective: The treatment of acute, complicated type B aortic dissection has evolved in the past several decades. Thoracic endovascular aortic repair when anatomy is suitable, has been regarded as the preferable treatment to seal the primary entry tear, redirect and re-establish adequate true lumen flow, and thereby promote aortic remodeling. This study was designed to determine the safety and efficacy of a conformable thoracic endoprosthesis device for patients with acute, complicated type B aortic dissection, defined as malperfusion or rupture or both.

Methods: Between January 2010 and January 2012, 50 patients with complicated type B aortic dissection from 26 sites in the United States were included in this prospective, multicenter, nonrandomized single-arm study. The primary safety end point was all-cause mortality through 30 days after treatment, and the primary efficacy end point was exclusion of the primary entry tear (Core Laboratory adjudicated) at 1-month follow-up. Secondary end points included false lumen thrombosis, dissection-based reintervention rate, and aortic rupture.

Results: All device implants were successfully completed. Six patients (12%) required additional device implantations ≤1 year from the index procedure. There was no conversion to open repair at 1 year. Exclusion of the primary entry tear at 30 days occurred in 97.5% of patients. All-cause mortality through 30 days was 8%. Survival was 88% at 1 year and 85% at 2 years. At 1 year after treatment, 35.1% of patients had experienced a decrease of ≥5 mm in overall diameter in the treated segment of the aorta. From pretreatment to the 36-month follow-up, the average minimum true lumen area increased by 206.3 mm(2), and the average maximum false lumen area decreased by 313.4 mm(2). The 30-day stroke rate was 18%; none were fatal, and one permanent deficit occurred. Four patients (8%) experienced spinal cord ischemia of any severity but without any permanent or significant deficits. New aortic dissection (3 retrograde, 2 de novo) occurred in five patients (10%). The secondary intervention rate was 18%.

Conclusions: Treatment with the conformable thoracic endovascular aortic repair device produced favorable perioperative and intermediate level clinical and anatomic outcomes. In particular, an operative mortality of 8% in this cohort is comparable to that noted in a Society for Vascular Surgery objective performance criteria publication. Late survival in our cohort compares favorably with historical data referable to complicated type B dissection.
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http://dx.doi.org/10.1016/j.jvs.2015.03.026DOI Listing
August 2015

Late aortic remodeling persists in the stented segment after endovascular repair of acute complicated type B aortic dissection.

J Vasc Surg 2015 Sep 6;62(3):600-5. Epub 2015 Jun 6.

Division of Vascular and Endovascular Surgery, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Mass.

Objective: Thoracic endovascular aortic repair (TEVAR) for acute complicated type B aortic dissection (AD) promotes early positive aortic remodeling. However, little is known about the long-term effect of TEVAR on the dissected aorta, which is the goal of this study.

Methods: Between August 2005 and August 2009, 31 patients with complicated type B AD were treated with TEVAR and had >1-year follow-up imaging. Computed tomography angiograms obtained at 1 month, 1 year, and long term (average, 42 months) were compared with baseline scans. The largest diameters of the stented thoracic aorta, stented true lumen, and stented false lumen were recorded at each time point, as were the values in the unstented distal thoracic aorta and the abdominal aorta. Changes over time were evaluated by a mixed effect analysis of variance model of repeated measures.

Results: The average age of the cohort was 56 years, and 74% were male. Indications for TEVAR were as follows: 61% malperfusion, 32% refractory hypertension, 45% impending rupture, and 32% persistent pain; 58% had more than one indication. All patients were treated in the acute phase within 7 days of the initial presentation. The average length of aorta covered was 19 cm. Observation of the stented segment over time showed that the maximum diameter of the stented thoracic aorta was stable (P = NS), the diameter of the stented true lumen increased (P < .001), and the diameter of the stented false lumen decreased (P < .001); 84% had complete false lumen obliteration across the stented aortic segment. Observation of the uncovered thoracic aorta over time showed that the maximum diameter increased (P = .014), as did the visceral segment of the aorta (P < .001). The average growth of the visceral segment was 31% in patients with a patent false lumen vs 3% in those with a thrombosed false lumen (P = .004). One patient had aneurysmal degeneration of the false lumen and required an additional endograft at 18 months.

Conclusions: TEVAR of acute AD promotes long-term remodeling across the stented segment, with false lumen obliteration in 84% of patients. However, false lumen obliteration beyond the stented segment appears necessary to prevent late aneurysmal degeneration of the distal aorta.
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http://dx.doi.org/10.1016/j.jvs.2015.03.064DOI Listing
September 2015

Cervical cancer screening coverage in a high-incidence region.

Rev Saude Publica 2015 27;49:17. Epub 2015 Feb 27.

OBJECTIVE To analyze the coverage of a cervical cancer screening program in a city with a high incidence of the disease in addition to the factors associated with non-adherence to the current preventive program. METHODS A cross-sectional study based on household surveys was conducted. The sample was composed of women between 25 and 59 years of age of the city of Boa Vista, RR, Northern Brazil who were covered by the cervical cancer screening program. The cluster sampling method was used. The dependent variable was participation in a women's health program, defined as undergoing at least one Pap smear in the 36 months prior to the interview; the explanatory variables were extracted from individual data. A generalized linear model was used. RESULTS 603 women were analyzed, with an mean age of 38.2 years (SD = 10.2). Five hundred and seventeen women underwent the screening test, and the prevalence of adherence in the last three years was up to 85.7% (95%CI 82.5;88.5). A high per capita household income and recent medical consultation were associated with the lower rate of not being tested in multivariate analysis. Disease ignorance, causes, and prevention methods were correlated with chances of non-adherence to the screening system; 20.0% of the women were reported to have undergone opportunistic and non-routine screening. CONCLUSIONS The informed level of coverage is high, exceeding the level recommended for the control of cervical cancer. The preventive program appears to be opportunistic in nature, particularly for the most vulnerable women (with low income and little information on the disease). Studies on the diagnostic quality of cervicovaginal cytology and therapeutic schedules for positive cases are necessary for understanding the barriers to the control of cervical cancer.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4386559PMC
http://dx.doi.org/10.1590/s0034-8910.2015049005554DOI Listing
July 2015

Controlling NMDA receptor subunit composition using ectopic retention signals.

J Neurosci 2014 Dec;34(50):16630-6

Ecole Normale Supérieure, Institut de Biologie de l'École Normale Supérieure, Paris F-75005, France, INSERM, U1024, Paris F-75005, France, and Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, Paris F-75005, France

Ligand-gated ion channels (LGICs) mediate fast synaptic transmission in the CNS. Typically, these membrane proteins are multimeric complexes associating several homologous subunits around a central pore. Because of the large repertoire of subunits within each family, LGICs exist in vivo as multiple subtypes that differ in subunit composition and functional properties. Establishing the specific properties of individual receptor subtypes remains a major goal in the field of neuroscience and molecular pharmacology. However, isolating specific receptor subtype in recombinant systems can be problematic because of the mixture of receptor populations. This is the case for NMDA receptors (NMDARs), a large family of tetrameric glutamate-gated ion channels that play key roles in brain physiology and pathology. A significant fraction of native NMDARs are triheteromers composed of two GluN1 subunits and two different GluN2 subunits (GluN2A-D). We developed a method based on dual retention signals adapted from G-protein-coupled GABA-B receptors allowing exclusive cell surface expression of triheteromeric rat NMDARs while coexpressed diheteromeric receptors (which contain a single type of GluN2 subunit) are retained intracellularly. Using this approach, we determined the functional properties of GluN1/GluN2A/GluN2B triheteromers, one of the most abundant NMDAR subtypes in the adult forebrain, revealing their unique gating and pharmacological attributes. We envision applicability of the retention signal approach for the study of a variety of heteromeric glutamate-gated ion channel receptors with defined subunit composition.
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http://dx.doi.org/10.1523/JNEUROSCI.2736-14.2014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6608501PMC
December 2014

Genetically encoding a light switch in an ionotropic glutamate receptor reveals subunit-specific interfaces.

Proc Natl Acad Sci U S A 2014 Apr 8;111(16):6081-6. Epub 2014 Apr 8.

Ecole Normale Supérieure, Institut de Biologie de l' Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale U1024, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8197, F-75005 Paris, France.

Reprogramming receptors to artificially respond to light has strong potential for molecular studies and interrogation of biological functions. Here, we design a light-controlled ionotropic glutamate receptor by genetically encoding a photoreactive unnatural amino acid (UAA). The photo-cross-linker p-azido-L-phenylalanine (AzF) was encoded in NMDA receptors (NMDARs), a class of glutamate-gated ion channels that play key roles in neuronal development and plasticity. AzF incorporation in the obligatory GluN1 subunit at the GluN1/GluN2B N-terminal domain (NTD) upper lobe dimer interface leads to an irreversible allosteric inhibition of channel activity upon UV illumination. In contrast, when pairing the UAA-containing GluN1 subunit with the GluN2A subunit, light-dependent inactivation is completely absent. By combining electrophysiological and biochemical analyses, we identify subunit-specific structural determinants at the GluN1/GluN2 NTD dimer interfaces that critically dictate UV-controlled inactivation. Our work reveals that the two major NMDAR subtypes differ in their ectodomain-subunit interactions, in particular their electrostatic contacts, resulting in GluN1 NTD coupling more tightly to the GluN2B NTD than to the GluN2A NTD. It also paves the way for engineering light-sensitive ligand-gated ion channels with subtype specificity through the genetic code expansion.
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http://dx.doi.org/10.1073/pnas.1318808111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000820PMC
April 2014

Expanding the genetic code in Xenopus laevis oocytes.

Chembiochem 2013 Jan 4;14(2):230-5. Epub 2013 Jan 4.

Ecole Normale Supérieure, Institut de Biologie de l'ENS, IBENS, 46 rue d'Ulm, Paris 75005, France.

Heterologous expression of ligand-gated ion channels (LGICs) in Xenopus laevis oocytes combined with site-directed mutagenesis has been demonstrated to be a powerful approach to study structure-function relationships. In particular, introducing unnatural amino acids (UAAs) has enabled modifications that are not found in natural proteins. However, the current strategy relies on the technically demanding in vitro synthesis of aminoacylated suppressor tRNA. We report here a general method that circumvents this limitation by utilizing orthogonal aminoacyl-tRNA synthetase (aaRS)/suppressor tRNA(CUA) pairs to genetically encode UAAs in Xenopus oocytes. We show that UAAs inserted in the N-terminal domain of N-methyl-D-aspartate receptors (NMDARs) serve as photo-crosslinkers that lock the receptor in a discrete conformational state in response to UV photo treatment. Our method should be generally applicable to studies of other LGICs in Xenopus oocytes.
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http://dx.doi.org/10.1002/cbic.201200515DOI Listing
January 2013

Zinc alleviates pain through high-affinity binding to the NMDA receptor NR2A subunit.

Nat Neurosci 2011 Jul 3;14(8):1017-22. Epub 2011 Jul 3.

Institut de Génétique et Biologie Moléculaire et Cellulaire, Illkirch, France.

Zinc is abundant in the central nervous system and regulates pain, but the underlying mechanisms are unknown. In vitro studies have shown that extracellular zinc modulates a plethora of signaling membrane proteins, including NMDA receptors containing the NR2A subunit, which display exquisite zinc sensitivity. We created NR2A-H128S knock-in mice to investigate whether Zn2+-NR2A interaction influences pain control. In these mice, high-affinity (nanomolar) zinc inhibition of NMDA currents was lost in the hippocampus and spinal cord. Knock-in mice showed hypersensitivity to radiant heat and capsaicin, and developed enhanced allodynia in inflammatory and neuropathic pain models. Furthermore, zinc-induced analgesia was completely abolished under both acute and chronic pain conditions. Our data establish that zinc is an endogenous modulator of excitatory neurotransmission in vivo and identify a new mechanism in pain processing that relies on NR2A NMDA receptors. The study also potentially provides a molecular basis for the pain-relieving effects of dietary zinc supplementation.
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http://dx.doi.org/10.1038/nn.2844DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4494785PMC
July 2011

Molecular basis of positive allosteric modulation of GluN2B NMDA receptors by polyamines.

EMBO J 2011 Jun 17;30(15):3134-46. Epub 2011 Jun 17.

Institut de Biologie de l'Ecole Normale Supérieure (IBENS), Ecole Normale Supérieure, CNRS UMR 8197, INSERM U1024, Paris, France.

NMDA receptors (NMDARs) form glutamate-gated ion channels that have central roles in neuronal communication and plasticity throughout the brain. Dysfunctions of NMDARs are involved in several central nervous system disorders, including stroke, chronic pain and schizophrenia. One hallmark of NMDARs is that their activity can be allosterically regulated by a variety of extracellular small ligands. While much has been learned recently regarding allosteric inhibition of NMDARs, the structural determinants underlying positive allosteric modulation of these receptors remain poorly defined. Here, we show that polyamines, naturally occurring polycations that selectively enhance NMDARs containing the GluN2B subunit, bind at a dimer interface between GluN1 and GluN2B subunit N-terminal domains (NTDs). Polyamines act by shielding negative charges present on GluN1 and GluN2B NTD lower lobes, allowing their close apposition, an effect that in turn prevents NTD clamshell closure. Our work reveals the mechanistic basis for positive allosteric modulation of NMDARs. It provides the first example of an intersubunit binding site in this class of receptors, a discovery that holds promise for future drug interventions.
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http://dx.doi.org/10.1038/emboj.2011.203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3160180PMC
June 2011

Subtle conformational changes between CX3CR1 genetic variants as revealed by resonance energy transfer assays.

FASEB J 2010 Nov 28;24(11):4585-98. Epub 2010 Jul 28.

Laboratoire Immunité et Infection, INSERM UMR-S 945, Paris, France.

The chemokine CX3CL1 is expressed as a membrane protein that forms a potent adhesive pair with its unique receptor CX3CR1. This receptor has 3 natural variants, V249-T280 (VT), I249-T280 (IT), and I249-M280 (IM), whose relative frequencies are significantly associated with the incidence of various inflammatory diseases. To assess the adhesive potency of CX3CR1 and the molecular diversity of its variants, we assayed their clustering status and their possible structural differences by fluorescence/bioluminescence resonance energy transfer (FRET or BRET) techniques. FRET assays by flow cytometry showed that the CX3CR1 variants cluster, in comparison with appropriate controls. BRET assays showed low nonspecific signals for VT and IT variants and high specific signals for IM, and thus pointed out a structural difference in this variant. We used molecular modeling to show how natural point mutations of CX3CR1 affect the packing of the 6th and 7th helices of this G-protein coupled receptor. Moreover, we found that the BRET technique is sensitive enough to detect these tiny changes. Consistently with our previous finding that CX3CL1 aggregates, our data here indicate that CX3CR1 clustering may contribute to the adhesiveness of the CX3CL1-CX3CR1 pair and may thus represent a new target for anti-inflammatory therapies.
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http://dx.doi.org/10.1096/fj.10-156612DOI Listing
November 2010

Functional evidence for a twisted conformation of the NMDA receptor GluN2A subunit N-terminal domain.

Neuropharmacology 2011 Jan 16;60(1):151-8. Epub 2010 Jul 16.

Institut de Biologie, CNRS UMR8197, INSERM U1024, Ecole Normale Supérieure, 46 rue d'Ulm, 75005 Paris, France.

Ionotropic glutamate receptors (iGluRs) possess in their extracellular region a large N-terminal domain (NTD) that precedes the agonist-binding domain and displays a clamshell-like architecture similar to the bacterial leucine/isoleucine/valine-binding protein (LIVBP). In addition to their role in receptor assembly, in NMDA receptors (NMDARs), the NTDs of GluN2A and GluN2B subunits form a major site for subunit-specific regulation of ion channel activity, in particular through binding of allosteric modulators such as the synaptically-enriched zinc ion. A recent crystallographic study of the isolated GluN2B NTD has revealed an unexpected twisted closed-cleft conformation caused by a rotation of ∼ 50° in the interlobe orientation compared with all other known LIVBP-like structures (Karakas et al., 2009). By measuring currents carried by recombinant NMDARs, we now provide functional evidence, through disulfide cross-linking and the identification of a new zinc-binding residue (D283), that the GluN2A NTD of intact GluN1/GluN2A receptors adopts a similar twisted conformation in its closed-cleft state. We propose that the twisted NTD conformation is a distinct structural feature of NMDARs (at least for GluN2A and GluN2B subunits), arguing for interactions between the NTDs in the tetrameric complex that are likely to differ between NMDA and AMPA/kainate receptors.
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http://dx.doi.org/10.1016/j.neuropharm.2010.07.003DOI Listing
January 2011

Unusual transfer of CutA into the secretory pathway, evidenced by fusion proteins with acetylcholinesterase.

FEBS J 2009 Aug 23;276(16):4473-82. Epub 2009 Jul 23.

Laboratoire de Neurobiologie, CNRS UMR 8544, Paris, France.

The mouse CutA protein exists as long and short components of 20 and 15 kDa, produced by the use of different in-frame ATGs initiation codons, and by proteolytic cleavage. We recently showed that, surprisingly, the longer, uncleaved component resides mostly in the secretory pathway and is secreted, whereas the shorter component resides mostly in the cytoplasm. To confirm these subcellular localizations, we constructed fusion proteins in which the catalytic domain of rat acetylcholinesterase was placed downstream of the CutA variants. The acquisition of an active conformation and N-glycosylation of the fusion proteins proved their transfer into the secretory pathway. We show that the CutA-AChE fusion proteins produced and secreted active, N-glycosylated molecules, while an AChE mutant lacking its secretory signal peptide did not produce any significant activity. Thus, an N-terminal CutA domain actually drives AChE into the endoplasmic reticulum and allows its secretion. This was observed with full length CutA, starting at Met1, and at a much lower level with the shorter mutants starting at Met24 and Met44, although the latter is not predicted to possess any signal peptide. These experiments illustrate the value of using AChE as a reporter and reveals an unusual protein trafficking and secretory process.
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http://dx.doi.org/10.1111/j.1742-4658.2009.07154.xDOI Listing
August 2009

Protein CutA undergoes an unusual transfer into the secretory pathway and affects the folding, oligomerization, and secretion of acetylcholinesterase.

J Biol Chem 2009 Feb 2;284(8):5195-207. Epub 2008 Dec 2.

Laboratoire de Neurobiologie, CNRS UMR 8544, Ecole Normale Supérieure, 46 Rue d'Ulm, 75005 Paris, France.

The mammalian protein CutA was first discovered in a search for the membrane anchor of mammalian brain acetylcholinesterase (AChE). It was co-purified with AChE, but it is distinct from the real transmembrane anchor protein, PRiMA. CutA is a ubiquitous trimeric protein, homologous to the bacterial CutA1 protein that belongs to an operon involved in resistance to divalent ions ("copper tolerance A"). The function of this protein in plants and animals is unknown, and several hypotheses concerning its subcellular localization have been proposed. We analyzed the expression and the subcellular localization of mouse CutA variants, starting at three in-frame ATG codons, in transfected COS cells. We show that CutA produces 20-kDa (H) and 15-kDa (L) components. The H component is transferred into the secretory pathway and secreted, without cleavage of a signal peptide, whereas the L component is mostly cytosolic. We show that expression of the longer CutA variant reduces the level of AChE, that this effect depends on the AChE C-terminal peptides, and probably results from misfolding. Surprisingly, CutA increased the secretion of a mutant possessing a KDEL motif at its C terminus; it also increased the formation of AChE homotetramers. We found no evidence for a direct interaction between CutA and AChE. The longer CutA variant seems to affect the processing and trafficking of secretory proteins, whereas the shorter one may have a distinct function in the cytoplasm.
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http://dx.doi.org/10.1074/jbc.M806260200DOI Listing
February 2009

Functional adhesiveness of the CX3CL1 chemokine requires its aggregation. Role of the transmembrane domain.

J Biol Chem 2008 Oct 25;283(44):30225-34. Epub 2008 Aug 25.

Laboratoire d'Immunologie Cellulaire, INSERM UMR-S 543, Université Pierre et Marie Curie-Paris 06, 91 boulevard de l'Hôpital, 75013 Paris, France.

In its native form, the chemokine CX3CL1 is a firmly adhesive molecule promoting leukocyte adhesion and migration and hence involved, along with its unique receptor CX3CR1, in various inflammatory processes. Here we investigated the role of molecular aggregation in the CX3CL1 adhesiveness. Assays of bioluminescence resonance energy transfer (BRET) and homogeneous time-resolved fluorescence (HTRF) in transfected cell lines and in primary cells showed specific signals indicative of CX3CL1 clustering. Truncation experiments showed that the transmembrane domain played a central role in this aggregation. A chimera with mutations of the 12 central transmembrane domain residues had significantly reduced BRET signals and characteristics of a non-clustering molecule. This mutant was weakly adhesive according to flow and dual pipette adhesion assays and was less glycosylated than CX3CL1, although, as we demonstrated, loss of glycosylation did not affect the CX3CL1 adhesive potency. We postulate that cell surfaces express CX3CL1 as a constitutive oligomer and that this oligomerization is essential for its adhesive potency. Inhibition of CX3CL1 self-assembly could limit the recruitment of CX3CR1-positive cells and may be a new pathway for anti-inflammatory therapies.
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http://dx.doi.org/10.1074/jbc.M802638200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2662081PMC
October 2008

Acetylcholinesterase associates differently with its anchoring proteins ColQ and PRiMA.

J Biol Chem 2008 Jul 29;283(30):20722-32. Epub 2008 May 29.

Laboratoire de Neurobiologie, Centre National de la Recherche Scientifique, UMR 8544, Ecole Normale Supérieure, 46 Rue d'Ulm, Paris, France.

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.
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http://dx.doi.org/10.1074/jbc.M801364200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258952PMC
July 2008
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