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1.
Immunity ; 57(7): 1696-1709.e10, 2024 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-38878770

RESUMO

Aicardi-Goutières syndrome (AGS) is an autoinflammatory disease characterized by aberrant interferon (IFN)-α production. The major cause of morbidity in AGS is brain disease, yet the primary source and target of neurotoxic IFN-α remain unclear. Here, we demonstrated that the brain was the primary source of neurotoxic IFN-α in AGS and confirmed the neurotoxicity of intracerebral IFN-α using astrocyte-driven Ifna1 misexpression in mice. Using single-cell RNA sequencing, we demonstrated that intracerebral IFN-α-activated receptor (IFNAR) signaling within cerebral endothelial cells caused a distinctive cerebral small vessel disease similar to that observed in individuals with AGS. Magnetic resonance imaging (MRI) and single-molecule ELISA revealed that central and not peripheral IFN-α was the primary determinant of microvascular disease in humans. Ablation of endothelial Ifnar1 in mice rescued microvascular disease, stopped the development of diffuse brain disease, and prolonged lifespan. These results identify the cerebral microvasculature as a primary mediator of IFN-α neurotoxicity in AGS, representing an accessible target for therapeutic intervention.


Assuntos
Encéfalo , Interferon-alfa , Microvasos , Malformações do Sistema Nervoso , Receptor de Interferon alfa e beta , Animais , Humanos , Camundongos , Interferon-alfa/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Receptor de Interferon alfa e beta/metabolismo , Receptor de Interferon alfa e beta/genética , Microvasos/patologia , Malformações do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/imunologia , Células Endoteliais/metabolismo , Camundongos Knockout , Masculino , Feminino , Transdução de Sinais , Camundongos Endogâmicos C57BL , Astrócitos/metabolismo , Modelos Animais de Doenças
2.
Proc Natl Acad Sci U S A ; 118(40)2021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34588305

RESUMO

Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.


Assuntos
Autofagia/fisiologia , Matriz Extracelular/patologia , Animais , Linhagem Celular , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Adesões Focais/metabolismo , Adesões Focais/patologia , Integrina alfaV/metabolismo , Camundongos , Células NIH 3T3 , Neoplasias/metabolismo , Neoplasias/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Células Estromais/metabolismo
3.
Cancer Cell Int ; 23(1): 49, 2023 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-36932402

RESUMO

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by an unfavorable prognosis for patients affected. During standard-of-care chemotherapy using temozolomide (TMZ), tumors acquire resistance thereby causing tumor recurrence. Thus, deciphering essential molecular pathways causing TMZ resistance are of high therapeutic relevance. METHODS: Mass spectrometry based proteomics were used to study the GBM proteome. Immunohistochemistry staining of human GBM tissue for either calpain-1 or -2 was performed to locate expression of proteases. In vitro cell based assays were used to measure cell viability and survival of primary patient-derived GBM cells and established GBM cell lines after TMZ ± calpain inhibitor administration. shRNA expression knockdowns of either calpain-1 or calpain-2 were generated to study TMZ sensitivity of the specific subunits. The Comet assay and É£H2AX signal measurements were performed in order to assess the DNA damage amount and recognition. Finally, quantitative real-time PCR of target proteins was applied to differentiate between transcriptional and post-translational regulation. RESULTS: Calcium-dependent calpain proteases, in particular calpain-2, are more abundant in glioblastoma compared to normal brain and increased in patient-matched initial and recurrent glioblastomas. On the cellular level, pharmacological calpain inhibition increased the sensitivities of primary glioblastoma cells towards TMZ. A genetic knockdown of calpain-2 in U251 cells led to increased caspase-3 cleavage and sensitivity to neocarzinostatin, which rapidly induces DNA strand breakage. We hypothesize that calpain-2 causes desensitization of tumor cells against TMZ by preventing strong DNA damage and subsequent apoptosis via post-translational TP53 inhibition. Indeed, proteomic comparison of U251 control vs. U251 calpain-2 knockdown cells highlights perturbed levels of numerous proteins involved in DNA damage response and downstream pathways affecting TP53 and NF-κB signaling. TP53 showed increased protein abundance, but no transcriptional regulation. CONCLUSION: TMZ-induced cell death in the presence of calpain-2 expression appears to favor DNA repair and promote cell survival. We conclude from our experiments that calpain-2 expression represents a proteomic mode that is associated with higher resistance via "priming" GBM cells to TMZ chemotherapy. Thus, calpain-2 could serve as a prognostic factor for GBM outcome.

4.
Int J Mol Sci ; 24(24)2023 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-38139457

RESUMO

The tumor microenvironment in glioblastoma (GB) is considered to be "cold", i.e., the fraction of cytotoxic T cells, for instance, is low. Instead, macrophages are the major immune cell population in GB, which stem either from tissue response (resident microglia) or recruitment of macrophages from the periphery, thereby undergoing tumor-dependent "imprinting" mechanisms by which macrophages can adapt a tumor-supportive phenotype. In this regard, it is important to describe the nature of macrophages associated with GB, in particular under therapy conditions using the gold standard chemotherapy drug temozolomide (TMZ). Here, we explored the suitability of combining information from in vivo magnetic resonance spectroscopic (MRS) approaches (metabolomics) with in vitro molecular analyses to assess therapy response and characterize macrophage populations in mouse GB using an isogenic GL261 model. For macrophage profiling, expression levels of matrix metalloproteinases (MMPs) and A disintegrin and metalloproteinases (ADAMs) were determined, since their gene products affect macrophage-tumor cell communication by extensive cleavage of immunomodulatory membrane proteins, such as PD-L1. In tumor mice with an overall therapy response, expression of genes encoding the proteases ADAM8, ADAM10, and ADAM17 was increased and might contribute to the immunosuppressive phenotype of GB and immune cells. In tumors responding to therapy, expression levels of ADAM8 were upregulated by TMZ, and higher levels of PD-L1 were correlated significantly. Using a CRISPR/Cas9 knockout of ADAM8 in GL261 cells, we demonstrated that soluble PD-L1 (sPD-L1) is only generated in the presence of ADAM8. Moreover, primary macrophages from WT and ADAM8-deficient mice showed ADAM8-dependent release of sPD-L1, independent of the macrophage polarization state. Since ADAM8 expression is induced in responding tumors and PD-L1 shedding is likely to decrease the anti-tumor activities of T-cells, we conclude that immunotherapy resistance is caused, at least in part, by the increased presence of proteases, such as ADAM8.


Assuntos
Glioblastoma , Glioma , Animais , Camundongos , Temozolomida/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/patologia , Antígeno B7-H1/metabolismo , Microambiente Tumoral/genética , Glioma/patologia , Linhagem Celular Tumoral
5.
Mol Cancer ; 21(1): 191, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36192757

RESUMO

BACKGROUND: In vivo gene editing of somatic cells with CRISPR nucleases has facilitated the generation of autochthonous mouse tumors, which are initiated by genetic alterations relevant to the human disease and progress along a natural timeline as in patients. However, the long and variable, orthotopic tumor growth in inner organs requires sophisticated, time-consuming and resource-intensive imaging for longitudinal disease monitoring and impedes the use of autochthonous tumor models for preclinical studies. METHODS: To facilitate a more widespread use, we have generated a reporter mouse that expresses a Cre-inducible luciferase from Gaussia princeps (GLuc), which is secreted by cells in an energy-consuming process and can be measured quantitatively in the blood as a marker for the viable tumor load. In addition, we have developed a flexible, complementary toolkit to rapidly assemble recombinant adenoviruses (AVs) for delivering Cre recombinase together with CRISPR nucleases targeting cancer driver genes. RESULTS: We demonstrate that intratracheal infection of GLuc reporter mice with CRISPR-AVs efficiently induces lung tumors driven by mutations in the targeted cancer genes and simultaneously activates the GLuc transgene, resulting in GLuc secretion into the blood by the growing tumor. GLuc blood levels are easily and robustly quantified in small-volume blood samples with inexpensive equipment, enable tumor detection already several months before the humane study endpoint and precisely mirror the kinetics of tumor development specified by the inducing gene combination. CONCLUSIONS: Our study establishes blood-based GLuc monitoring as an inexpensive, rapid, high-throughput and animal-friendly method to longitudinally monitor autochthonous tumor growth in preclinical studies.


Assuntos
Copépodes , Neoplasias Pulmonares , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Copépodes/genética , Copépodes/metabolismo , Edição de Genes , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Neoplasias Pulmonares/genética , Camundongos
6.
Brain Behav Immun ; 101: 194-210, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35032575

RESUMO

BACKGROUND: Antigen-specific neuroinflammation and neurodegeneration are characteristic for neuroimmunological diseases. In Parkinson's disease (PD) pathogenesis, α-synuclein is a known culprit. Evidence for α-synuclein-specific T cell responses was recently obtained in PD. Still, a causative link between these α-synuclein responses and dopaminergic neurodegeneration had been lacking. We thus addressed the functional relevance of α-synuclein-specific immune responses in PD in a mouse model. METHODS: We utilized a mouse model of PD in which an Adeno-associated Vector 1/2 serotype (AAV1/2) expressing human mutated A53T-α-Synuclein was stereotactically injected into the substantia nigra (SN) of either wildtype C57BL/6 or Recombination-activating gene 1 (RAG1)-/- mice. Brain, spleen, and lymph node tissues from different time points following injection were then analyzed via FACS, cytokine bead assay, immunohistochemistry and RNA-sequencing to determine the role of T cells and inflammation in this model. Bone marrow transfer from either CD4+/CD8-, CD4-/CD8+, or CD4+/CD8+ (JHD-/-) mice into the RAG-1-/- mice was also employed. In addition to the in vivo studies, a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay was utilized. RESULTS: AAV-based overexpression of pathogenic human A53T-α-synuclein in dopaminergic neurons of the SN stimulated T cell infiltration. RNA-sequencing of immune cells from PD mouse brains confirmed a pro-inflammatory gene profile. T cell responses were directed against A53T-α-synuclein-peptides in the vicinity of position 53 (68-78) and surrounding the pathogenically relevant S129 (120-134). T cells were required for α-synuclein-induced neurodegeneration in vivo and in vitro, while B cell deficiency did not protect from dopaminergic neurodegeneration. CONCLUSIONS: Using T cell and/or B cell deficient mice and a newly developed A53T-α-synuclein-expressing neuronal cell culture/immune cell assay, we confirmed in vivo and in vitro that pathogenic α-synuclein peptide-specific T cell responses can cause dopaminergic neurodegeneration and thereby contribute to PD-like pathology.


Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Modelos Animais de Doenças , Dopamina , Neurônios Dopaminérgicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Doença de Parkinson/patologia , RNA , Substância Negra/metabolismo , Linfócitos T/metabolismo , alfa-Sinucleína/metabolismo
7.
Proc Natl Acad Sci U S A ; 116(17): 8397-8402, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30962377

RESUMO

Actin filaments (F-actin) are key components of sarcomeres, the basic contractile units of skeletal muscle myofibrils. A crucial step during myofibril differentiation is the sequential exchange of α-actin isoforms from smooth muscle (α-SMA) and cardiac (α-CAA) to skeletal muscle α-actin (α-SKA) that, in mice, occurs during early postnatal life. This "α-actin switch" requires the coordinated activity of actin regulators because it is vital that sarcomere structure and function are maintained during differentiation. The molecular machinery that controls the α-actin switch, however, remains enigmatic. Cyclase-associated proteins (CAP) are a family of actin regulators with largely unknown physiological functions. We here report a function for CAP2 in regulating the α-actin exchange during myofibril differentiation. This α-actin switch was delayed in systemic CAP2 mutant mice, and myofibrils remained in an undifferentiated stage at the onset of the often excessive voluntary movements in postnatal mice. The delay in the α-actin switch coincided with the onset of motor function deficits and histopathological changes including a high frequency of type IIB ring fibers. Our data suggest that subtle disturbances of postnatal F-actin remodeling are sufficient for predisposing muscle fibers to form ring fibers. Cofilin2, a putative CAP2 interaction partner, has been recently implicated in myofibril actin cytoskeleton differentiation, and the myopathies in cofilin2 and CAP2 mutant mice showed striking similarities. We therefore propose a model in which CAP2 and cofilin2 cooperate in actin regulation during myofibril differentiation.


Assuntos
Citoesqueleto de Actina/fisiologia , Proteínas de Transporte , Diferenciação Celular , Músculo Esquelético , Miofibrilas/fisiologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Knockout , Desenvolvimento Muscular/genética , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/fisiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Biochem Biophys Res Commun ; 567: 215-221, 2021 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-34171798

RESUMO

Neuroblastoma (NB), a pediatric cancer of the peripheral sympathetic nervous system, represents the most frequent solid malignancy in infants. Treatment of high-risk patients is still challenging and, depending on the genetic make-up and involved risk factors, the 5-year survival rate can drop to only 30%. Here, we found that the expression of the Dual Specificity Tyrosine Phosphorylation Regulated Kinase 3 (DYRK3) is increased in NB and is associated with decreased survival in NB patients. We further identified DYRK3 as a cytoplasmic kinase in NB cells and found that its levels are increased by hypoxic conditions. Further mechanistic studies revealed that DYRK3 acts as a negative regulator of HIF-driven transcriptional responses, suggesting that it functions in a negative feedback loop controlling the hypoxic response. Moreover, DYRK3 negatively impacted on NB cell differentiation, proposing an oncogenic role of this kinase in the etiology of NB. In summary, we describe novel functions of the DYRK3 kinase in NB, which will help to further improve the understanding of this disease eventually leading to the design of improved therapeutic concepts.


Assuntos
Neuroblastoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Neuroblastoma/patologia , Proteínas Serina-Treonina Quinases/análise , Proteínas Tirosina Quinases/análise , Hipóxia Tumoral
9.
Biol Chem ; 402(2): 195-206, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33544472

RESUMO

Glioblastoma multiforme (GBM) is the most aggressive type of brain cancer with a median survival of only 15 months. To complement standard treatments including surgery, radiation and chemotherapy, it is essential to understand the contribution of the GBM tumor microenvironment. Brain macrophages and microglia particularly contribute to tumor angiogenesis, a major hallmark of GBM. ADAM8, a metalloprotease-disintegrin strongly expressed in tumor cells and associated immune cells of GBMs, is related to angiogenesis and correlates with poor clinical prognosis. However, the specific contribution of ADAM8 to GBM tumorigenesis remains elusive. Knockdown of ADAM8 in U87 glioma cells led to significantly decreased angiogenesis and tumor volumes of these cells after stereotactic injection into striate body of mice. We found that the angiogenic potential of ADAM8 in GBM cells and in primary macrophages is mediated by the regulation of osteopontin (OPN), an important inducer of tumor angiogenesis. By in vitro cell signaling analyses, we demonstrate that ADAM8 regulates OPN via JAK/STAT3 pathway in U87 cells and in primary macrophages. As ADAM8 is a dispensable protease for physiological homeostasis, we conclude that ADAM8 could be a tractable target to modulate angiogenesis in GBM with minor side-effects.


Assuntos
Proteínas ADAM/metabolismo , Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Patológica/metabolismo , Osteopontina/metabolismo , Proteínas ADAM/deficiência , Proteínas ADAM/genética , Animais , Neoplasias Encefálicas/patologia , Proliferação de Células , Células Cultivadas , Glioblastoma/patologia , Humanos , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neovascularização Patológica/patologia
10.
J Med Genet ; 57(3): 212-216, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31446422

RESUMO

BACKGROUND: Cerebral cavernous malformations (CCMs) can cause severe neurological morbidity but our understanding of the mechanisms that drive CCM formation and growth is still incomplete. Recent experimental data suggest that dysfunctional CCM3-deficient endothelial cell clones form cavernous lesions in conjunction with normal endothelial cells. OBJECTIVE: In this study, we addressed the question whether endothelial cell mosaicism can be found in human cavernous tissue of CCM1 germline mutation carriers. METHODS AND RESULTS: Bringing together single-molecule molecular inversion probes in an ultra-sensitive sequencing approach with immunostaining to visualise the lack of CCM1 protein at single cell resolution, we identified a novel late postzygotic CCM1 loss-of-function variant in the cavernous tissue of a de novo CCM1 germline mutation carrier. The extended unilateral CCM had been located in the right central sulcus causing progressive proximal paresis of the left arm at the age of 15 years. Immunohistochemical analyses revealed that individual caverns are lined by both heterozygous (CCM1+/- ) and compound heterozygous (CCM1-/- ) endothelial cells. CONCLUSION: We here demonstrate endothelial cell mosaicism within single caverns of human CCM tissue. In line with recent in vitro data on CCM1-deficient endothelial cells, our results provide further evidence for clonal evolution in human CCM1 pathogenesis.


Assuntos
Predisposição Genética para Doença , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Proteína KRIT1/genética , Mosaicismo , Adolescente , Proteínas Reguladoras de Apoptose/genética , Proteínas de Transporte/genética , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Mutação em Linhagem Germinativa/genética , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas Proto-Oncogênicas/genética , Zigoto/patologia
11.
Int J Mol Sci ; 23(1)2021 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-35008590

RESUMO

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Anidrases Carbônicas/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Temozolomida/farmacologia , Antineoplásicos Alquilantes/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Glioblastoma/metabolismo , Humanos , Recidiva Local de Neoplasia/tratamento farmacológico , Recidiva Local de Neoplasia/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais/efeitos dos fármacos
12.
Neurogenetics ; 19(1): 55-59, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29197946

RESUMO

Familial cerebral cavernous malformations (CCMs) predispose to seizures and hemorrhagic stroke. Molecular genetic analyses of CCM1, CCM2, and CCM3 result in a mutation detection rate of up to 98%. However, only whole genome sequencing (WGS) in combination with the Manta algorithm for analyses of structural variants revealed a heterozygous 24 kB inversion including exon 1 of CCM2 in a 12-year-old boy with familial CCMs. Its breakpoints were fine-mapped, and quantitative analysis on RNA confirmed reduced CCM2 expression. Our data expand the spectrum of CCM mutations and indicate that the existence of a fourth CCM disease gene is rather unlikely.


Assuntos
Proteínas de Transporte/genética , Inversão Cromossômica , Hemangioma Cavernoso do Sistema Nervoso Central/genética , Encéfalo/diagnóstico por imagem , Encéfalo/patologia , Criança , Estudo de Associação Genômica Ampla , Hemangioma Cavernoso do Sistema Nervoso Central/diagnóstico por imagem , Hemangioma Cavernoso do Sistema Nervoso Central/patologia , Humanos , Masculino , Linhagem , Sequenciamento Completo do Genoma
13.
Int J Cancer ; 142(4): 779-791, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28986926

RESUMO

Metastatic breast cancer affects long-term survival and is a major cause of cancer death for women worldwide. The Metalloprotease-Disintegrin ADAM8 promotes breast cancer development and brain metastasis in a mouse breast cancer model. Here, abundant ADAM8 expression was detected in primary human breast tumors and associated brain metastases. To investigate the function of ADAM8 in metastasis, MB-231 breast cancer cells with ADAM8 knockdown (MB-231_shA8) and scramble control cells (MB-231_shCtrl) were analyzed for their capability to develop metastases. In vitro, formation of metastatic complexes in hanging drops is dependent on ADAM8 and blocked by ADAM8 inhibition. MB-231_shA8 in contrast to MB-231_shCtrl cells were impaired in transmigration through an endothelial and a reconstituted blood-brain barrier. Out of 23 MMP and 22 ADAM genes, only the MMP-9 gene was affected by ADAM8 knockdown in MB-231_shA8 cells. Following re-expression of wild-type ADAM8 in contrast to ADAM8 lacking the cytoplasmic domain in MB-231_shA8 cells caused increased levels of activated pERK1/2 and pCREB (S133) that were associated with elevated MMP-9 transcription. Application of ADAM8 and MMP-9 antibodies reduced transmigration of MB-231 cells suggesting that ADAM8 affects transmigration of breast cancer cells by MMP-9 regulation. ADAM8-dependent transmigration was confirmed in Hs578t cells overexpressing ADAM8. Moreover, transmigration of MB-231 and Hs578t cells was significantly reduced for cells treated with an antibody directed against P-selectin glycoprotein ligand (PSGL-1), a substrate of ADAM8. From these data we conclude that ADAM8 promotes early metastatic processes such as transendothelial migration by upregulation of MMP-9 and shedding of PSGL-1 from breast cancer cells.


Assuntos
Proteínas ADAM/biossíntese , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Células Endoteliais/patologia , Metaloproteinase 9 da Matriz/biossíntese , Proteínas de Membrana/biossíntese , Proteínas ADAM/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/genética , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Técnicas de Cocultura , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Metaloproteinase 9 da Matriz/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Pessoa de Meia-Idade
14.
Histopathology ; 68(5): 657-65, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26249211

RESUMO

AIMS: Evaluation of intraepidermal nerve fibres (IENFs) in skin biopsies is used in the diagnosis of small-fibre neuropathies. The number of IENFs is assessed manually under a microscope, with an inter-rater variability of ~25%. Unless the images are digitized, there is no documentation. Our aim was to develop a method for standardized semi-automated quantification (SAQ) and documentation of IENF density. METHODS AND RESULTS: We analysed samples from four different university centres that were immunostained according to local protocols. Images were acquired through the Z-plane with a whole slide scanner. orbit image analysis software was used to create an analysable image and develop a reliable algorithm for IENF detection. Rebuilt images revealed well-contrasted nerves, allowing detection of IENFs (automated). The software presented the detected nerves for confirmation by the operator (manual). As compared with the conventional microscopy count, the SAQ achieved correlation coefficients of 0.99 and 0.96 and interfacility variabilities of 19% and 23%, respectively. We found better reproducibility with fluorescence-stained specimens than with bright-field images. CONCLUSIONS: The new semi-automated method has high experimenter-independent reproducibility when based on nerve detection by fluorescence and is easy to perform, even by untrained users. The IENF counting is electronically well documented.


Assuntos
Fibras Nervosas/patologia , Pele/inervação , Automação Laboratorial , Biópsia , Humanos , Processamento de Imagem Assistida por Computador , Reprodutibilidade dos Testes
15.
J Magn Reson Imaging ; 39(3): 665-72, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23723131

RESUMO

PURPOSE: To assess ultrasmall superparamagnetic iron oxide particles (USPIO) -enhanced MR imaging for the differentiation of malignant from benign, inflammatory lesions. MATERIALS AND METHODS: In this study, approved by the local animal care committee, VX2 carcinoma and intramuscular abscesses were implanted into the hind thighs of New Zealand White rabbits. MR imaging was performed pre contrast and serially for 24 h after the injection of USPIO. MR findings were compared with histopathologic results based on Prussian blue stains for the presence of iron. RESULTS: Twenty-four hours after the Ferumoxtran-injection, no changes were observed in VX2 carcinomas, whereas a mean reduction of the contrast-to-noise ratio (CNR) of approximately 90% was noticed in abscesses as well as in necrotic tumors. On histopathologic examination, abscess and necrotic parts of the tumor were found to include iron-containing monocytes demonstrating that the reduction in CNR was caused by USPIO-tagged monocytes. CONCLUSION: Our results prove the ability of USPIO-enhanced MRI to differentiate benign, inflammatory from malignant lesions.


Assuntos
Abscesso/patologia , Dextranos , Imageamento por Ressonância Magnética/métodos , Nanopartículas de Magnetita , Neoplasias/patologia , Abscesso/diagnóstico , Animais , Biópsia por Agulha , Diagnóstico Diferencial , Modelos Animais de Doenças , Xenoenxertos , Humanos , Imuno-Histoquímica , Inflamação/diagnóstico , Inflamação/patologia , Masculino , Transplante de Neoplasias , Neoplasias/diagnóstico , Coelhos , Distribuição Aleatória
16.
Brain Commun ; 6(1): fcae017, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38317856

RESUMO

The immunoproteasome is a central protease complex required for optimal antigen presentation. Immunoproteasome activity is also associated with facilitating the degradation of misfolded and oxidized proteins, which prevents cellular stress. While extensively studied during diseases with increasing evidence suggesting a role for the immunoproteasome during pathological conditions including neurodegenerative diseases, this enzyme complex is believed to be mainly not expressed in the healthy brain. In this study, we show an age-dependent increase in polyubiquitination in the brains of wild-type mice, accompanied by an induction of immunoproteasomes, which was most prominent in neurons and microglia. In contrast, mice completely lacking immunoproteasomes (triple-knockout mice), displayed a strong increase in polyubiquitinated proteins already in the young brain and developed spontaneous epileptic seizures, beginning at the age of 6 months. Injections of kainic acid led to high epilepsy-related mortality of aged triple-knockout mice, confirming increased pathological hyperexcitability states. Notably, the expression of the immunoproteasome was reduced in the brains of patients suffering from epilepsy. In addition, the aged triple-knockout mice showed increased anxiety, tau hyperphosphorylation and degeneration of Purkinje cell population with the resulting ataxic symptoms and locomotion alterations. Collectively, our study suggests a critical role for the immunoproteasome in the maintenance of a healthy brain during ageing.

17.
Cells ; 13(7)2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38607071

RESUMO

Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.


Assuntos
Amidas , Antineoplásicos , Compostos Bicíclicos Heterocíclicos com Pontes , Glioblastoma , Pirimidinas , Sulfonamidas , Animais , Camundongos , Temozolomida/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Metotrexato/farmacologia , Metotrexato/uso terapêutico , Citarabina/farmacologia , Citarabina/uso terapêutico , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Apoptose
18.
Neurol Res Pract ; 6(1): 19, 2024 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-38570823

RESUMO

OBJECTIVE: Brain tumors and metastases account for approximately 10% of all status epilepticus (SE) cases. This study described the clinical characteristics, treatment, and short- and long-term outcomes of this population. METHODS: This retrospective, multi-center cohort study analyzed all brain tumor patients treated for SE at the university hospitals of Frankfurt and Marburg between 2011 and 2017. RESULTS: The 208 patients (mean 61.5 ± 14.7 years of age; 51% male) presented with adult-type diffuse gliomas (55.8%), metastatic entities (25.5%), intracranial extradural tumors (14.4%), or other tumors (4.3%). The radiological criteria for tumor progression were evidenced in 128 (61.5%) patients, while 57 (27.4%) were newly diagnosed with tumor at admission and 113 (54.3%) had refractory SE. The mean hospital length of stay (LOS) was 14.8 days (median 12.0, range 1-57), 171 (82.2%) patients required intensive care (mean LOS 8.9 days, median 5, range 1-46), and 44 (21.2%) were administered mechanical ventilation. All patients exhibited significant functional status decline (modified Rankin Scale) post-SE at discharge (p < 0.001). Mortality at discharge was 17.3% (n = 36), with the greatest occurring in patients with metastatic disease (26.4%, p = 0.031) and those that met the radiological criteria for tumor progression (25%, p < 0.001). Long-term mortality at one year (65.9%) was highest in those diagnosed with adult-type diffuse gliomas (68.1%) and metastatic disease (79.2%). Refractory status epilepticus cases showed lower survival rates than non-refractory SE patients (log-rank p = 0.02) and those with signs of tumor progression (log-rank p = 0.001). CONCLUSIONS: SE occurrence contributed to a decline in functional status in all cases, regardless of tumor type, tumor progression status, and SE refractoriness, while long-term mortality was increased in those with malignant tumor entities, tumor progressions, and refractory SE. SE prevention may preserve functional status and improve survival in individuals with brain tumors.

19.
EMBO Rep ; 12(2): 129-35, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21212807

RESUMO

Adaptor proteins allow temporal and spatial coordination of signalling. In this study, we show SUMOylation of the adaptor protein TANK and its interacting kinase TANK-binding kinase 1 (TBK1). Modification of TANK by the small ubiquitin-related modifier (SUMO) at the evolutionarily conserved Lys 282 is triggered by the kinase activities of IκB kinase ɛ (IKKɛ) and TBK1. Stimulation of TLR7 leads to inducible SUMOylation of TANK, which in turn weakens the interaction with IKKɛ and thus relieves the negative function of TANK on signal propagation. Reconstitution experiments show that an absence of TANK SUMOylation impairs inducible expression of distinct TLR7-dependent target genes, providing a molecular mechanism that allows the control of TANK function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína SUMO-1/metabolismo , Receptor 7 Toll-Like/metabolismo , Cisteína Endopeptidases , Endopeptidases/metabolismo , Células HEK293 , Humanos , Quinase I-kappa B/metabolismo , Fosforilação , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais
20.
Clin Neuropathol ; 32(4): 286-90, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23320996

RESUMO

Multiple system atrophy (MSA) is a rapidly progressive sporadic α-synucleinopathy with adult onset characterized by progressive cerebellar ataxia, basal ganglia symptoms, autonomic dysfunction and pyramidal tract signs. While full-blown dementia is considered an exclusion criterion according to Consensus Guidelines, mild cognitive deficits such as fronto-executive dysfunction have been reported in some MSA individuals. However, the underlying anatomic correlate still has to be elucidated. We here report a 74-year-old patient with a clinical diagnosis of "probable MSA of the cerebellar type (MSA-C)" who developed pronounced clinical symptoms of fronto-executive dysfunction. Neuropathologic investigations revealed (1) numerous glial cytoplasmic inclusions (GCI) in the putamen, mesencephalon and cerebellum, (2) pronounced betaamyloid pathology in the frontal lobe and (3) mild hippocampal τ-pathology. In this patient, fronto-executive dysfunction can easily be explained by frontal degeneration typical for AD. These findings challenge the concept of cognitive dysfunction as a core feature of MSA as long as concomitant pathology other than MSA has not been reliably excluded by post mortem analysis.


Assuntos
Peptídeos beta-Amiloides/análise , Atrofia de Múltiplos Sistemas/patologia , Proteínas tau/análise , Idoso , Peptídeos beta-Amiloides/metabolismo , Humanos , Masculino , Atrofia de Múltiplos Sistemas/metabolismo , alfa-Sinucleína/análise , alfa-Sinucleína/biossíntese , Proteínas tau/biossíntese
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