Leonor Miller-Fleming

Publications (5) View all

• Article: Visualization of cell-to-cell transmission of mutant huntingtin oligomers.

  Federico Herrera, Sandra Tenreiro, Leonor Miller-Fleming, Tiago Fleming Outeiro
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  ABSTRACT: We developed a new cell model for the visualization of toxic huntingtin oligomers in living cells. Huntingtin exon 1 (25Q or 103Q) was fused to non-fluorescent halves of the Venus protein. When huntingtin dimerizes inside the cells, Venus becomes functionally reconstituted and emits fluorescence. Oligomerization, aggregation and toxicity of mutant huntingtin were assessed by several procedures. We also present evidence that the transmission of huntingtin between cells can be determined in a quantitative manner with our model. Thus, this model can be a powerful screening tool for the identification of modifiers of oligomerization and cell-to-cell traffic of mutant huntingtin.
  PLoS currents. 01/2011; 3:RRN1210.
• Article: Functional gene expression profiling in yeast implicates translational dysfunction in mutant huntingtin toxicity.

  Eran Tauber, Leonor Miller-Fleming, Robert P Mason, Wanda Kwan, Jannine Clapp, Nicola J Butler, Tiago F Outeiro, Paul J Muchowski, Flaviano Giorgini
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  ABSTRACT: Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.
  Journal of Biological Chemistry 11/2010; 286(1):410-9. · 4.77 Impact Factor
• Article: Novel SPG3A and SPG4 mutations in dominant spastic paraplegia families.

  J L Loureiro, L Miller-Fleming, C Thieleke-Matos, P Magalhães, V T Cruz, P Coutinho, J Sequeiros, I Silveira
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  ABSTRACT: The hereditary spastic paraplegias (HSP) are a genetically and clinically heterogeneous group of neurodegenerative disorders, mainly characterized by a progressive spasticity and weakness of the lower limbs. Mutations in the SPG4 and SPG3A genes are responsible for approximately 50% of autosomal dominant HSP. To genetically diagnose the Portuguese families with HSP, mutation analysis was performed for the SPG4 and SPG3A genes. Analysis was performed by polymerase chain reaction, followed by denaturing high performance liquid chromatography (DHPLC), in 61 autosomal dominant (AD)-HSP families and 19 unrelated patients without family history. Ten novel mutations were identified: one in the SPG3A and nine in the SPG4 genes; three known mutations in the SPG4 were also found. Most of the novel mutations were frameshift or nonsense (80%), resulting in a dysfunctional protein. The SPG4 and SPG3A analysis allowed the identification of 10 novel mutations and the genetic diagnosis of approximately a quarter of our AD-HSP families.
  Acta Neurologica Scandinavica 08/2008; 119(2):113-8. · 2.47 Impact Factor
• Article: Autosomal Dominant Spastic Paraplegias: A Review of 89 Families Resulting From a Portuguese Survey.

  José Leal Loureiro, Eva Brandão, Luis Ruano, Ana F Brandão, Ana M Lopes, Carolina Thieleke-Matos, Leonor Miller-Fleming, Vitor T Cruz, Mafalda Barbosa, Isabel Silveira, Giovanni Stevanin, Jorge Pinto-Basto, Jorge Sequeiros, Isabel Alonso, Paula Coutinho
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  ABSTRACT: IMPORTANCE Hereditary spastic paraplegias (HSPs) are a group of diseases caused by corticospinal tract degeneration. Mutations in 3 genes (SPG4, SPG3, and SPG31) are said to be the cause in half of the autosomal dominant HSPs (AD-HSPs). This study is a systematic review of families with HSP resulting from a population-based survey. Novel genotype-phenotype correlations were established. OBJECTIVE To describe the clinical, genetic, and epidemiological features of Portuguese AD-HSP families. DESIGN Retrospective medical record review. SETTING A population-based systematic survey of hereditary ataxias and spastic paraplegias conducted in Portugal from 1993 to 2004. PARTICIPANTS Families with AD-HSP. MAIN OUTCOME MEASURE Mutation detection in the most prevalent genes. RESULTS We identified 239 patients belonging to 89 AD-HSP families. The prevalence was 2.4 in 100 000. Thirty-one distinct mutations (26 in SPG4, 4 in SPG3, and 1 in SPG31) segregated in 41% of the families (33.7%, 6.2%, and 1.2% had SPG4, SPG3 and SPG31 mutations, respectively). Seven of the SPG4 mutations were novel, and 7% of all SPG4 mutations were deletions. When disease onset was before the first decade, 31% had SPG4 mutations and 27% had SPG3 mutations. In patients with SPG4 mutations, those with large deletions had the earliest disease onset, followed by those with missense, frameshift, nonsense, and alternative-splicing mutations. Rate of disease progression was not significantly different among patients with SPG3 and SPG4 mutations in a multivariate analysis. For patients with SPG4 mutations, disease progression was worst in patients with later-onset disease. CONCLUSIONS AND RELEVANCE The prevalence of AD-HSP and frequency of SPG3 and SPG4 mutations in the current study were similar to what has been described in other studies except that the frequency of SPG4 deletions was lower. In contrast, the frequency of SPG31 mutations in the current study was rare compared with other studies. The most interesting aspects of this study are that even in patients with early-onset disease the probability of finding a SPG4 mutation was higher than for patients with SPG3 mutations; there was no difference in disease progression with genotype but an association with the age at onset; 7 new SPG4 mutations were identified; and for the first time, to our knowledge, the nature of the SPG4 mutations was found to predict the age at onset.
  JAMA neurology. 02/2013;
• Source

  Available from: Flaviano Giorgini

  Article: Yeast as a model for studying human neurodegenerative disorders.

  Leonor Miller-Fleming, Flaviano Giorgini, Tiago F Outeiro
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  ABSTRACT: Protein misfolding and aggregation are central events in many disorders including several neurodegenerative diseases. This suggests that alterations in normal protein homeostasis may contribute to pathogenesis, but the exact molecular mechanisms involved are still poorly understood. The budding yeast Saccharomyces cerevisiae is one of the model systems of choice for studies in molecular medicine. Modeling human neurodegenerative diseases in this simple organism has already shown the incredible power of yeast to unravel the complex mechanisms and pathways underlying these pathologies. Indeed, this work has led to the identification of several potential therapeutic targets and drugs for many diseases, including the neurodegenerative diseases. Several features associated with these diseases, such as formation of protein aggregates, cellular toxicity mediated by misfolded proteins, oxidative stress and hallmarks of apoptosis have been faithfully recapitulated in yeast, enabling researchers to take advantage of this powerful model to rapidly perform genetic and compound screens with the aim of identifying novel candidate therapeutic targets and drugs. Here we review the work undertaken to model human brain disorders in yeast, and how these models provide insight into novel therapeutic approaches for these diseases.
  Biotechnology Journal 04/2008; 3(3):325-38.