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1.
Nat Commun ; 12(1): 5074, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417463

RESUMO

ß cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human ß cells with inflammation but its expression is reduced in surviving ß cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate ß cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO ß cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between ß cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in ß cells may reduce activating pathologic immune cells and killing of ß cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 1/patologia , Inflamação/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Citotoxicidade Imunológica , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Feminino , Humanos , Imunidade , Inflamação/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Transcrição Genética
2.
Vaccines (Basel) ; 9(8)2021 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-34451982

RESUMO

Despite limited data on safety and immunogenicity, heterologous prime-boost vaccination is currently recommended for individuals with ChAdOx1 nCoV-19 prime immunization in certain age groups. In this prospective, single-center study we included 166 health care workers from Heidelberg University Hospital who received either heterologous ChAdOx1 nCoV-19/BNT162b2, homologous BNT162b2 or homologous ChAdOx1 nCoV-19 vaccination between December 2020 and May 2021. We measured anti-S1 IgG, SARS-CoV-2 specific neutralizing antibodies, and antibodies against different SARS-CoV-2 fragments 0-3 days before and 19-21 days after boost vaccination. Before boost, 55/70 (79%) ChAdOx1 nCoV-19-primed compared with 44/45 (98%) BNT162b2-primed individuals showed positive anti-S1 IgG with a median (IQR) anti-S1 IgG index of 1.95 (1.05-2.99) compared to 9.38 (6.26-17.12). SARS-CoV-2 neutralizing antibodies exceeded the threshold in 24/70 (34%) of ChAdOx1 nCoV-19-primed and 43/45 (96%) of BNT162b2-primed individuals. After boosting dose, median (IQR) anti-S1 IgG index in heterologous ChAdOx1 nCoV-19/BNT162b2 vaccinees was 116.2 (61.84-170), compared to 13.09 (7.03-29.02) in homologous ChAdOx1 nCoV-19 and 145.5 (100-291.1) in homologous BNT162b2 vaccinees. All boosted vaccinees exceeded the threshold for neutralization, irrespective of their vaccination scheme. Vaccination was well-tolerated overall. We show that heterologous ChAdOx1 nCoV-19/BNT162b2 vaccination is safe and induces a strong and broad humoral response in healthy individuals.

3.
Clin J Am Soc Nephrol ; 16(7): 1073-1082, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34031181

RESUMO

BACKGROUND AND OBJECTIVES: Patients receiving hemodialysis are at high risk for both severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and severe coronavirus disease 2019. A lifesaving vaccine is available, but sensitivity to vaccines is generally lower in patients on dialysis. Little is yet known about antibody responses after coronavirus disease 2019 (COVID-19) vaccination in this vulnerable group. DESIGN, SETTING, PARTICIPANTS, AND MEASUREMENTS: In this prospective single-center study, we included 22 patients on dialysis and 46 healthy controls from Heidelberg University Hospital between December 2020 and February 2021. We measured anti-S1 IgG with a threshold index for detection greater than one, neutralizing antibodies with a threshold for viral neutralization of ≥30%, and antibodies against different SARS-CoV2 fragments 17-22 days after the first dose and 18-22 days after the second dose of the mRNA vaccine BNT162b2. RESULTS: After the first vaccine dose, four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls developed positive anti-S1 IgG, with a median anti-S1 IgG index of 0.2 (interquartile range, 0.1-0.7) compared with nine (interquartile range, 4-16), respectively. SARS-CoV2 neutralizing antibodies exceeded the threshold for neutralization in four of 22 (18%) patients on dialysis compared with 43 of 46 (93%) healthy controls, with a median percent inhibition of 11 (interquartile range, 3-24) compared with 65 (interquartile range, 49-75), respectively. After the second dose, 14 of 17 (82%) patients on dialysis developed neutralizing antibodies exceeding the threshold for viral neutralization and antibodies against the receptor binding S1 domain of the spike protein, compared with 46 of 46 (100%) healthy controls, respectively. The median percent inhibition was 51 (interquartile range, 32-86) compared with 98 (interquartile range, 97-98) in healthy controls. CONCLUSIONS: Patients receiving long-term hemodialysis show a reduced antibody response to the first and second doses of the mRNA vaccine BNT162b2. The majority (82%) develop neutralizing antibodies after the second dose but at lower levels than healthy controls.


Assuntos
Anticorpos Antivirais/sangue , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , Diálise Renal , SARS-CoV-2/imunologia , Vacinação , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade
4.
Ann Clin Transl Neurol ; 8(4): 877-886, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33704933

RESUMO

BACKGROUND: Inflammation in chronic active lesions occurs behind a closed blood-brain barrier and cannot be detected with MRI. Activated microglia are highly enriched for iron and can be visualized with quantitative susceptibility mapping (QSM), an MRI technique used to delineate iron. OBJECTIVE: To characterize the histopathological correlates of different QSM hyperintensity patterns in MS lesions. METHODS: MS brain slabs were imaged with MRI and QSM, and processed for histology. Immunolabeled cells were quantified in the lesion rim, center, and adjacent normal-appearing white matter (NAWM). Iron+ myeloid cell densities at the rims were correlated with susceptibilities. Human-induced pluripotent stem cell (iPSC)-derived microglia were used to determine the effect of iron on the production of reactive oxygen species (ROS) and pro-inflammatory cytokines. RESULTS: QSM hyperintensity at the lesion perimeter correlated with activated iron+ myeloid cells in the rim and NAWM. Lesions with high punctate or homogenous QSM signal contained no or minimally activated iron- myeloid cells. In vitro, iron accumulation was highest in M1-polarized human iPSC-derived microglia, but it did not enhance ROS or cytokine production. CONCLUSION: A high QSM signal outlining the lesion rim but not punctate signal in the center is a biomarker for chronic inflammation in white matter lesions.

5.
Artigo em Inglês | MEDLINE | ID: mdl-32269065

RESUMO

OBJECTIVE: To identify coinhibitory immune pathways important in the brain, we hypothesized that comparison of T cells in lesions from patients with MS with tumor-infiltrating T cells (TILs) from patients with glioblastoma multiforme may reveal novel targets for immunotherapy. METHODS: We collected fresh surgical resections and matched blood from patients with glioblastoma, blood and unmatched postmortem CNS tissue from patients with MS, and blood from healthy donors. The expression of TIGIT, CD226, and their shared ligand CD155 as well as PD-1 and PDL1 was assessed by both immunohistochemistry and flow cytometry. RESULTS: We found that TIGIT was highly expressed on glioblastoma-infiltrating T cells, but was near-absent from MS lesions. Conversely, lymphocytic expression of PD-1/PD-L1 was comparable between the 2 diseases. Moreover, TIGIT was significantly upregulated in circulating lymphocytes of patients with glioblastoma compared with healthy controls, suggesting recirculation of TILs. Expression of CD226 was also increased in glioblastoma, but this costimulatory receptor was expressed alongside TIGIT in the majority of tumor-infiltrating T cells, suggesting functional counteraction. CONCLUSIONS: The opposite patterns of TIGIT expression in the CNS between MS and glioblastoma reflects the divergent features of the immune response in these 2 CNS diseases. These data raise the possibility that anti-TIGIT therapy may be beneficial for patients with glioblastoma.

6.
Acta Neuropathol Commun ; 7(1): 130, 2019 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-31405387

RESUMO

Activated myeloid cells and astrocytes are the predominant cell types in active multiple sclerosis (MS) lesions. Both cell types can adopt diverse functional states that play critical roles in lesion formation and resolution. In order to identify phenotypic subsets of myeloid cells and astrocytes, we profiled two active MS lesions with thirteen glial activation markers using imaging mass cytometry (IMC), a method for multiplexed labeling of histological sections. In the acutely demyelinating lesion, we found multiple distinct myeloid and astrocyte phenotypes that populated separate lesion zones. In the post-demyelinating lesion, phenotypes were less distinct and more uniformly distributed. In both lesions cell-to-cell interactions were not random, but occurred between specific glial subpopulations and lymphocytes. Finally, we demonstrated that myeloid, but not astrocyte phenotypes were activated along a lesion rim-to-center gradient, and that marker expression in glial cells at the lesion rim was driven more by cell-extrinsic factors than in cells at the center. This proof-of-concept study demonstrates that highly multiplexed tissue imaging, combined with the appropriate computational tools, is a powerful approach to study heterogeneity, spatial distribution and cellular interactions in the context of MS lesions. Identifying glial phenotypes and their interactions at different lesion stages may provide novel therapeutic targets for inhibiting acute demyelination and low-grade, chronic inflammation.


Assuntos
Astrócitos/patologia , Comunicação Celular/fisiologia , Esclerose Múltipla Recidivante-Remitente/patologia , Células Mieloides/patologia , Fenótipo , Adulto , Astrócitos/metabolismo , Feminino , Humanos , Masculino , Esclerose Múltipla Recidivante-Remitente/metabolismo , Células Mieloides/metabolismo
8.
Nat Commun ; 9(1): 5337, 2018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30559390

RESUMO

Epigenetic annotation studies of genetic risk variants for multiple sclerosis (MS) implicate dysfunctional lymphocytes in MS susceptibility; however, the role of central nervous system (CNS) cells remains unclear. We investigated the effect of the risk variant, rs7665090G, located near NFKB1, on astrocytes. We demonstrated that chromatin is accessible at the risk locus, a prerequisite for its impact on astroglial function. The risk variant was associated with increased NF-κB signaling and target gene expression, driving lymphocyte recruitment, in cultured human astrocytes and astrocytes within MS lesions, and with increased lesional lymphocytic infiltrates and lesion sizes. Thus, our study establishes a link between genetic risk for MS (rs7665090G) and dysfunctional astrocyte responses associated with increased CNS access for peripheral immune cells. MS may therefore result from variant-driven dysregulation of the peripheral immune system and of the CNS, where perturbed CNS cell function aids in establishing local autoimmune inflammation.


Assuntos
Astrócitos/metabolismo , Sistema Nervoso Central/citologia , Esclerose Múltipla/genética , Subunidade p50 de NF-kappa B/genética , Células Cultivadas , Sistema Nervoso Central/patologia , Predisposição Genética para Doença/genética , Humanos , Esclerose Múltipla/patologia , Subunidade p50 de NF-kappa B/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Fatores de Risco , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
9.
Front Immunol ; 9: 217, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29515568

RESUMO

The role traditionally assigned to astrocytes in the pathogenesis of multiple sclerosis (MS) lesions has been the formation of the glial scar once inflammation has subsided. Astrocytes are now recognized to be early and highly active players during lesion formation and key for providing peripheral immune cells access to the central nervous system. Here, we review the role of astrocytes in the formation and evolution of MS lesions, including the recently described functional polarization of astrocytes, discuss prototypical pathways for astrocyte activation, and summarize mechanisms by which MS treatments affect astrocyte function.


Assuntos
Astrócitos/imunologia , Barreira Hematoencefálica/imunologia , Sistema Nervoso Central/imunologia , Imunossupressores/uso terapêutico , Esclerose Múltipla/imunologia , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Barreira Hematoencefálica/citologia , Barreira Hematoencefálica/efeitos dos fármacos , Sistema Nervoso Central/citologia , Sistema Nervoso Central/patologia , Humanos , Imunossupressores/farmacologia , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/patologia , Transdução de Sinais/imunologia
10.
J Magn Reson Imaging ; 48(5): 1281-1287, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-29517817

RESUMO

BACKGROUND: The pathological processes in the first weeks of multiple sclerosis (MS) lesion formation include myelin digestion that breaks chemical bonds in myelin lipid layers. This can increase lesion magnetic susceptibility, which is a potentially useful biomarker in MS patient management, but not yet investigated. PURPOSE: To understand and quantify the effects of myelin digestion on quantitative susceptibility mapping (QSM) of MS lesions. STUDY TYPE: Histological and QSM analyses on in vitro models of myelin breakdown and MS lesion formation in vivo. POPULATION/SPECIMENS: Acutely demyelinating white matter lesions from MS autopsy tissue were stained with the lipid dye oil red O. Myelin basic protein (MBP), a major membrane protein of myelin, was digested with trypsin. Purified human myelin was denatured with sodium dodecyl sulfate (SDS). QSM was performed on phantoms containing digestion products and untreated controls. In vivo QSM was performed on five MS patients with newly enhancing lesions, and then repeated within 2 weeks. FIELD STRENGTH/SEQUENCE: 3D T 2 * -weighted spoiled multiecho gradient echo scans performed at 3T. ASSESSMENT: Region of interest analyses were performed by a biochemist and a neuroradiologist to determine susceptibility changes on in vitro and in vivo QSM images. STATISTICAL TESTS: Not applicable. RESULTS: MBP degradation by trypsin increased the QSM measurement by an average of 112 ± 37 ppb, in excellent agreement with a theoretical estimate of 111 ppb. Degradation of human myelin by SDS increased the QSM measurement by 23 ppb. As MS lesions changed from gadolinium enhancing to nonenhancing over an average of 15.8 ± 3.7 days, their susceptibility increased by an average of 7.5 ± 6.3 ppb. DATA CONCLUSION: Myelin digestion in the early stages of MS lesion formation contributes to an increase in tissue susceptibility, detectable by QSM, as a lesion evolves from gadolinium enhancing to nonenhancing. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 3 J. Magn. Reson. Imaging 2018;47:1281-1287.


Assuntos
Imageamento por Ressonância Magnética , Esclerose Múltipla/diagnóstico por imagem , Bainha de Mielina/química , Algoritmos , Animais , Autopsia , Biomarcadores/química , Bovinos , Humanos , Proteína Básica da Mielina/química , Imagens de Fantasmas , Tripsina/química , Substância Branca/diagnóstico por imagem
11.
Ann Neurol ; 83(1): 131-141, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29283442

RESUMO

OBJECTIVE: Myeloid cells, including macrophages and dendritic cells, are a prominent component of central nervous system (CNS) infiltrates during multiple sclerosis (MS) and the animal model experimental autoimmune encephalomyelitis (EAE). Although myeloid cells are generally thought to be proinflammatory, alternatively polarized subsets can serve noninflammatory and/or reparative functions. Here we investigate the heterogeneity and biological properties of myeloid cells during central nervous system autoimmunity. METHODS: Myeloid cell phenotypes in chronic active MS lesions were analyzed by immunohistochemistry. In addition, immune cells were isolated from the CNS during exacerbations and remissions of EAE and characterized by flow cytometric, genetic, and functional assays. RESULTS: Myeloid cells expressing inducible nitric oxide synthase (iNOS), indicative of a proinflammatory phenotype, were detected in the actively demyelinating rim of chronic active MS lesions, whereas macrophages expressing mannose receptor (CD206), a marker of alternatively polarized human myeloid cells, were enriched in the quiescent lesion core. During EAE, CNS-infiltrating myeloid cells, as well as microglia, shifted from expression of proinflammatory markers to expression of noninflammatory markers immediately prior to clinical remissions. Murine CNS myeloid cells expressing the alternative lineage marker arginase-1 (Arg1) were partially derived from iNOS+ precursors and were deficient in activating encephalitogenic T cells compared with their Arg1- counterparts. INTERPRETATION: These observations demonstrate the heterogeneity of CNS myeloid cells, their evolution during the course of autoimmune demyelinating disease, and their plasticity on the single cell level. Future therapeutic strategies for disease modification in individuals with MS may be focused on accelerating the transition of CNS myeloid cells from a proinflammatory to a noninflammatory phenotype. Ann Neurol 2018;83:131-141.


Assuntos
Doenças Autoimunes do Sistema Nervoso/patologia , Plasticidade Celular , Células Mieloides/patologia , Animais , Arginase/genética , Células da Medula Óssea/patologia , Quimera , Progressão da Doença , Encefalomielite Autoimune Experimental/patologia , Humanos , Imuno-Histoquímica , Lectinas Tipo C/biossíntese , Lectinas Tipo C/genética , Lectinas de Ligação a Manose/biossíntese , Lectinas de Ligação a Manose/genética , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/patologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Fenótipo , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/genética
12.
JCI Insight ; 2(17)2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28878118

RESUMO

Recent data indicate that there are different subpopulations of Th17 cells that can express a regulatory as opposed to an inflammatory gene signature. The transmembrane glycoprotein PDPN is critical in the development of multiple organs including the lymphatic system and has been described on T cells in mouse models of autoimmune Th17 inflammation. Here, we demonstrate that unlike in mice, PDPN+ T cells induced under classic Th17-polarizing conditions express transcription factors associated with Th17 cells but do not produce IL-17. Moreover, these cells express a transcriptional profile enriched for immunosuppressive and regulatory pathways and express a distinct cytokine profile compared with potentially pathogenic PDPN- Th17 cells. Ligation of PDPN by its ligand CLEC-2 ameliorates the Th17 inflammatory response. IL-17 secretion is restored with shRNA gene silencing of PDPN. Furthermore, PDPN expression is reduced via an Sgk1-mediated pathway under proinflammatory, high sodium chloride conditions. Finally, CD3+PDPN+ T cells are devoid of IL-17 in skin biopsies from patients with candidiasis, a prototypical Th17-driven skin disease. Thus, our data support the hypothesis that PDPN may serve as a marker of a nonpathogenic Th17 cell subset and may also functionally regulate pathogenic Th17 inflammation.


Assuntos
Inflamação/patologia , Glicoproteínas de Membrana/fisiologia , Células Th17/patologia , Células Cultivadas , Citocinas/metabolismo , Perfilação da Expressão Gênica , Inativação Gênica , Humanos , Proteínas Imediatamente Precoces/metabolismo , Interleucina-17/biossíntese , Lectinas Tipo C/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/genética , Células Th17/metabolismo , Transcrição Genética
13.
Brain ; 140(2): 399-413, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28007993

RESUMO

Astrocytes are key players in the pathology of multiple sclerosis and can assume beneficial and detrimental roles during lesion development. The triggers and timing of the different astroglial responses in acute lesions remain unclear. Astrocytes in acute multiple sclerosis lesions have been shown previously to contain myelin debris, although its significance has not been examined. We hypothesized that myelin phagocytosis by astrocytes is an early event during lesion formation and leads to astroglial immune responses. We examined multiple sclerosis lesions and other central nervous system pathologies with prominent myelin injury, namely, progressive multifocal leukoencephalopathy, metachromatic leukodystrophy and subacute infarct. In all conditions, we found that myelin debris was present in most astrocytes at sites of acute myelin breakdown, indicating that astroglial myelin phagocytosis is an early and prominent feature. Functionally, myelin debris was taken up by astrocytes through receptor-mediated endocytosis and resulted in astroglial NF-κB activation and secretion of chemokines. These in vitro results in rats were validated in human disease where myelin-positive hypertrophic astrocytes showed increased nuclear localization of NF-κB and elevated chemokine expression compared to myelin-negative, reactive astrocytes. Thus, our data suggest that myelin uptake is an early response of astrocytes in diseases with prominent myelin injury that results in recruitment of immune cells. This first line response of astrocytes to myelin injury may exert beneficial or detrimental effects on the lesion pathology, depending on the inflammatory context. Modulating this response might be of therapeutic relevance in multiple sclerosis and other demyelinating conditions.


Assuntos
Astrócitos/metabolismo , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/patologia , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Fagocitose/fisiologia , Adulto , Idoso , Animais , Animais Recém-Nascidos , Astrócitos/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Pré-Escolar , Cultura , Citocinas/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Feminino , Humanos , Hidrazonas/farmacologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/patologia , Fatores de Tempo , Fator de Crescimento Transformador beta/farmacologia
14.
Front Cell Neurosci ; 10: 33, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27013967

RESUMO

BACKGROUND: Schizophrenia can be conceptualized as a form of dysconnectivity between brain regions.To investigate the neurobiological foundation of dysconnectivity, one approach is to analyze white matter structures, such as the pathology of fiber tracks. S100B is considered a marker protein for glial cells, in particular oligodendrocytes and astroglia, that passes the blood brain barrier and is detectable in peripheral blood. Earlier Studies have consistently reported increased S100B levels in schizophrenia. In this study, we aim to investigate associations between S100B and structural white matter abnormalities. METHODS: We analyzed data of 17 unmedicated schizophrenic patients (first and recurrent episode) and 22 controls. We used voxel based morphometry (VBM) to detect group differences of white matter structures as obtained from T1-weighted MR-images and considered S100B serum levels as a regressor in an age-corrected interaction analysis. RESULTS: S100B was increased in both patient subgroups. Using VBM, we found clusters indicating significant differences of the association between S100B concentration and white matter. Involved anatomical structures are the posterior cingulate bundle and temporal white matter structures assigned to the superior longitudinal fasciculus. CONCLUSIONS: S100B-associated alterations of white matter are shown to be existent already at time of first manifestation of psychosis and are distinct from findings in recurrent episode patients. This suggests involvement of S100B in an ongoing and dynamic process associated with structural brain changes in schizophrenia. However, it remains elusive whether increased S100B serum concentrations in psychotic patients represent a protective response to a continuous pathogenic process or if elevated S100B levels are actively involved in promoting structural brain damage.

16.
J Biol Chem ; 287(27): 23119-27, 2012 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-22593584

RESUMO

Nicotinic acetylcholine receptor (nAChR) cell surface expression levels are modulated during nicotine dependence and multiple disorders of the nervous system, but the mechanisms underlying nAChR trafficking remain unclear. To determine the role of cysteine residues, including their palmitoylation, on neuronal α4 nAChR subunit maturation and cell surface trafficking, the cysteines in the two intracellular regions of the receptor were replaced with serines using site-directed mutagenesis. Palmitoylation is a post-translational modification that regulates membrane receptor trafficking and function. Metabolic labeling with [(3)H]palmitate determined that the cysteine in the cytoplasmic loop between transmembrane domains 1 and 2 (M1-M2) is palmitoylated. When this cysteine is mutated to a serine, producing a depalmitoylated α4 nAChR, total protein expression decreases, but surface expression increases compared with wild-type α4 levels, as determined by Western blotting and enzyme-linked immunoassays, respectively. The cysteines in the M3-M4 cytoplasmic loop do not appear to be palmitoylated, but replacing all of the cysteines in the loop with serines increases total and cell surface expression. When all of the intracellular cysteines in both loops are mutated to serines, there is no change in total expression, but there is an increase in surface expression. Calcium accumulation assays and high affinity binding for [(3)H]epibatidine determined that all mutants retain functional activity. Thus, our results identify a novel palmitoylation site on cysteine 273 in the M1-M2 loop of the α4 nAChR and determine that cysteines in both intracellular loops are regulatory factors in total and cell surface protein expression of the α4ß2 nAChR.


Assuntos
Lipoilação/fisiologia , Neurônios/fisiologia , Transporte Proteico/fisiologia , Receptores Nicotínicos/metabolismo , Animais , Cálcio/metabolismo , Técnicas de Cocultura , Cisteína/genética , Citoplasma/metabolismo , Células HEK293 , Hipocampo/citologia , Humanos , Neurônios/citologia , Palmitatos/metabolismo , Palmitatos/farmacologia , Cultura Primária de Células , Estrutura Terciária de Proteína , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Trítio
17.
Am J Med Genet B Neuropsychiatr Genet ; 153B(1): 291-7, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-19330775

RESUMO

Several lines of evidence suggest an important role of the S100B protein and its coding gene in different neuropathological and psychiatric disorders like dementia, bipolar affective disorders and schizophrenia. To clarify whether a direct link exists between gene and gene product, that is, whether S100B variants directly modulate S100B serum concentration, 196 healthy individuals were assessed for S100B serum concentrations and genotyped for five potentially functional S100B SNPs. Functional variants of the serotonergic genes 5-HT1A and 5-HTT possibly modulating S100B serum levels were also studied. Further, publicly available human postmortem gene expression data were re-analyzed to elucidate the impact of S100B, 5-HT1A and 5-HTT SNPs on frontal cortex S100B mRNA expression. Several S100B SNPs, particularly rs9722, and the S100B haplotype T-G-G-A (including rs2186358-rs11542311-rs2300403-rs9722) were associated with elevated S100B serum concentrations (Bonferroni corrected P < 0.05). Of these, rs11542311 was also associated with S100B mRNA expression directly (Bonferroni corrected P = 0.05) and within haplotype G-A-T-C (rs11542311-rs2839356-rs9984765-rs881827; P = 0.004), again with the G-allele increasing S100B expression. Our results suggest an important role of S100B SNPs on S100B serum concentrations and S100B mRNA expression. It hereby links recent evidence for both, the impact of S100B gene variation on various neurological or psychiatric disorders like dementia, bipolar affective disorders and schizophrenia and the strong relation between S100B serum levels and these disorders.


Assuntos
Fatores de Crescimento Neural/genética , Proteínas S100/genética , Humanos , Fatores de Crescimento Neural/sangue , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética , Receptor 5-HT1A de Serotonina/genética , Fatores de Risco , Subunidade beta da Proteína Ligante de Cálcio S100 , Proteínas S100/sangue , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
18.
J Geriatr Psychiatry Neurol ; 22(1): 23-34, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19196630

RESUMO

The association between cytokines (IL-1 beta, sIL-4R, IL-6, IL-8, IL-10, IL-12, TNF-alpha) and subcortical white matter lesions, cortical atrophy and lacunar infarctions of the aging brain was investigated among 268 elderly community participants. Single pro- and anti-inflammatory cytokines were neither associated with WML nor with atrophy and lacunar infarction. An association between atrophy and the chemokine-cytokine factor (containing sIL-4R, IL-6, IL-8) remained significant after adjustment for age, gender, education, depressive symptoms, diabetes mellitus, cardiovascular diseases (stroke, TIA, myocardial infarction, myocardial insufficiency, arrhythmic heart), hypertension, body-mass index, smoking status and aggregation inhibitors as opposed to single cytokines. Atrophy of the parietal, temporal and occipital lobes was associated with the same cytokine-chemokine factor for both the whole sample or restricted to those without history of stroke/TIA. The results indicate that a combination of chemokine-cytokines rather than single cytokines may contribute to inflammatory processes associated with cortical atrophy in the aging brain.


Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Citocinas/sangue , Imageamento por Ressonância Magnética/métodos , Distribuição por Idade , Idoso , Atrofia/diagnóstico , Atrofia/epidemiologia , Encéfalo/patologia , Infarto Encefálico/diagnóstico , Infarto Encefálico/epidemiologia , Comorbidade , Estudos Transversais , Diabetes Mellitus/diagnóstico , Diabetes Mellitus/epidemiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Alemanha/epidemiologia , Humanos , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Interleucinas/sangue , Masculino , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/epidemiologia , Razão de Chances , Análise de Componente Principal , Distribuição por Sexo , Acidente Vascular Cerebral/diagnóstico , Acidente Vascular Cerebral/epidemiologia
19.
Prog Neuropsychopharmacol Biol Psychiatry ; 32(8): 1789-92, 2008 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-18718498

RESUMO

Astrocyte activation indicated by increased S100B is considered a potential pathogenic factor for schizophrenia. To investigate the relationship between astrocyte activation and cognitive performance, S100B serum concentration, memory performance, and psychopathology were assessed in 40 first-episode and 35 chronic schizophrenia patients upon admission and after four weeks of treatment. Chronic schizophrenia patients with high S100B were impaired concerning verbal memory performance (AVLT, Auditory Verbal Learning Test) compared to chronic and first-episode patients with low S100B levels. The findings support the hypothesis that astrocyte activation might contribute to the development of cognitive dysfunction in schizophrenia.


Assuntos
Transtornos da Memória/sangue , Transtornos da Memória/etiologia , Fatores de Crescimento Neural/sangue , Proteínas S100/sangue , Esquizofrenia/complicações , Adulto , Doença Crônica , Feminino , Humanos , Masculino , Testes Neuropsicológicos , Subunidade beta da Proteína Ligante de Cálcio S100 , Esquizofrenia/sangue , Aprendizagem Verbal/fisiologia , Adulto Jovem
20.
Neurosci Lett ; 442(2): 100-3, 2008 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-18638525

RESUMO

The mainly glia-derived protein S100B has been shown to be involved in the pathophysiology of diseases such as neurodegenerative diseases, schizophrenia or depression. These diseases go along with distinct changes of cerebral neurotransmitters and neurotrophic factors. Few and partly inconsistent data exist on the influence of cerebral S100B protein levels on different neurotransmitters. Therefore we investigated levels of serotonin (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA), noradrenaline (NA), dopamine (DA), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the hippocampus, frontal cortex and residual neocortex in S100B knock out (S100B KO) mice compared to wildtype controls. There was a significant increase of hippocampal BDNF (+53%) and a decrease of hippocampal (-12%) and residual neocortical (-15%) NA in 10-month-old S100B KO mice compared to wildtype mice whereas the other mediators investigated did not show genotype-dependent changes. The increased hippocampal BDNF may represent an endogenous attempt to compensate trophic effects of S100B protein especially on serotonergic neurons, which have been shown to be unaffected in S100B KO mice previously. As referred to changes in NA levels functional studies are warranted to elucidate the link between S100B protein and the noradrenergic metabolism.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/genética , Fatores de Crescimento Neural/deficiência , Norepinefrina/metabolismo , Proteínas S100/deficiência , Animais , Monoaminas Biogênicas/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Camundongos , Camundongos Knockout , Subunidade beta da Proteína Ligante de Cálcio S100 , Serotonina/metabolismo
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