Marianna Alunni-Fabbroni

Publications of Marianna Alunni-Fabbroni

• A high-throughput DNA methylation analysis of a single cell.

  Authors: Martin Kantlehner, Roland Kirchner, Petra Hartmann, Joachim W Ellwart, Marianna Alunni-Fabbroni, Axel Schumacher

  Nucleic acids research. 01/2011; 39(7):e44.

  In recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of beingIn recent years, the field of epigenetics has grown dramatically and has become one of the most dynamic and fast-growing branches of molecular biology. The amount of diseases suspected of being influenced by DNA methylation is rising steadily and includes common diseases such as schizophrenia, bipolar disorder, Alzheimer's disease, diabetes, atherosclerosis, cancer, major psychosis, lupus and Parkinson's disease. Due to cellular heterogeneity of methylation patterns, epigenetic analyses of single cells become a necessity. One rationale is that DNA methylation profiles are highly variable across individual cells, even in the same organ, dependent on the function of the gene, disease state, exposure to environmental factors (e.g. radiation, drugs or nutrition), stochastic fluctuations and various other causes. Using a polymerase chain reaction (PCR)-slide microreaction system, we present here a methylation-sensitive PCR analysis, the restriction enzyme-based single-cell methylation assay (RSMA), in the analysis of DNA methylation patterns in single cells. This method addresses the problems of cell heterogeneity in epigenetics research; it is comparably affordable, avoids complicated microfluidic systems and offers the opportunity for high-throughput screening, as many single cells can be screened in parallel. In addition to this study, critical principles and caveats of single cell methylation analyses are discussed.

• Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6.

  Authors: Fabienne C Fiesel, Aaron Voigt, Stephanie S Weber, Chris Van den Haute, Andrea Waldenmaier, Karin Görner, Michael Walter, Marlene L Anderson, Jeannine V Kern, Tobias M Rasse, Thorsten Schmidt, Wolfdieter Springer, Roland Kirchner, Michael Bonin, Manuela Neumann, Veerle Baekelandt, Marianna Alunni-Fabbroni, Jörg B Schulz, Philipp J Kahle

  The EMBO journal. 11/2009;

  TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis.TDP-43 is an RNA/DNA-binding protein implicated in transcriptional repression and mRNA processing. Inclusions of TDP-43 are hallmarks of frontotemporal dementia and amyotrophic lateral sclerosis. Besides aggregation of TDP-43, loss of nuclear localization is observed in disease. To identify relevant targets of TDP-43, we performed expression profiling. Thereby, histone deacetylase 6 (HDAC6) downregulation was discovered on TDP-43 silencing and confirmed at the mRNA and protein level in human embryonic kidney HEK293E and neuronal SH-SY5Y cells. This was accompanied by accumulation of the major HDAC6 substrate, acetyl-tubulin. HDAC6 levels were restored by re-expression of TDP-43, dependent on RNA binding and the C-terminal protein interaction domains. Moreover, TDP-43 bound specifically to HDAC6 mRNA arguing for a direct functional interaction. Importantly, in vivo validation in TDP-43 knockout Drosophila melanogaster confirmed the specific downregulation of HDAC6. HDAC6 is necessary for protein aggregate formation and degradation. Indeed, HDAC6-dependent reduction of cellular aggregate formation and increased cytotoxicity of polyQ-expanded ataxin-3 were found in TDP-43 silenced cells. In conclusion, loss of functional TDP-43 causes HDAC6 downregulation and might thereby contribute to pathogenesis.

• Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

  Authors: Thomas Kroneis, Liat Gutstein-Abo, Kristina Kofler, Michaele Hartmann, Petra Hartmann, Marianna Alunni-Fabbroni, Wolfgang Walcher, Gottfried Dohr, Erwin Petek, Esther Guetta, Peter Sedlmayr

  Journal of cellular and molecular medicine. 06/2009;

  ABSTRACT The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosisABSTRACT The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis since it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser-catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested by using samples containing mixed cells of related and non-related individuals. Single cell DNA fingerprinting was successful in 74% of the cells analyzed (55/74) with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single cell basis.

• Regulation of astrocyte inflammatory responses by the Parkinson's disease-associated gene DJ-1.

  Authors: Jens Waak, Stephanie S Weber, Andrea Waldenmaier, Karin Görner, Marianna Alunni-Fabbroni, Heinrich Schell, Daniela Vogt-Weisenhorn, Thu-Trang Pham, Veerle Reumers, Veerle Baekelandt, Wolfgang Wurst, Philipp J Kahle

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 03/2009;

  The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulatesThe Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.-Waak, J., Weber, S. S., Waldenmaier, A., Görner, K., Alunni-Fabbroni, M., Schell, H., Vogt-Weisenhorn, D., Pham, T.-T., Reumers, V., Baekelandt, V., Wurst, W., Kahle, P. J. Regulation of astrocyte inflammatory responses by the Parkinson's disease-associated gene DJ-1.

• Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

  Authors: Fabienne C Fiesel, Aaron Voigt, Stephanie S Weber, Chris Van den Haute, Andrea Waldenmaier, Karin GÖRner, Michael Walter, Marlene L Anderson, Jeannine V Kern, Tobias M Rasse, Thorsten Schmidt, Wolfdieter Springer, Roland Kirchner, Michael Bonin, Manuela Neumann, Veerle Baekelandt, Marianna Alunni-Fabbroni, Jörg B Schulz, Philipp J Kahle

  The EMBO Journal. 29(1):209-221.

  The EMBO Journal 29, 209 (2009). doi:10.1038/emboj.2009.324

• Knockdown of transactive response DNA-binding protein (TDP-43) downregulates histone deacetylase 6

  Authors: Fabienne C Fiesel, Aaron Voigt, Stephanie S Weber, Chris Van den Haute, Andrea Waldenmaier, Karin GÖRner, Michael Walter, Marlene L Anderson, Jeannine V Kern, Tobias M Rasse, Thorsten Schmidt, Wolfdieter Springer, Roland Kirchner, Michael Bonin, Manuela Neumann, Veerle Baekelandt, Marianna Alunni-Fabbroni, Jörg B Schulz, Philipp J Kahle

  The EMBO Journal. 29(1):209-221.

  The EMBO Journal 29, 209 (2009). doi:10.1038/emboj.2009.324