- Email: [email protected]
Expression profiling and bioinformatics analysis of dysregulated microRNAs (miRNAs) in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)
S20
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
glutamatergic pyramidal cell populations were quantitatively assessed. The degree of subsequent neuronal loss was also determined. Results: Quantitative quadruple immunofluorescence revealed reduced expression of mitochondrial respiratory chain complexes I and IV within GABAergic interneurons; indicating mitochondrial dysfunction in the parietal tissue of Alpers’ patients. Relative densities of GABAergic interneurons and pyramidal cells were reduced. Mislocalisation of GABAergic interneurons but not pyramidal cells was observed in the white matter of patient parietal tissue. Conclusions: We propose that respiratory deficiencies combined with loss of GABAergic interneurons and pyramidal cells leads to an imbalance between inhibition and excitation, which may contribute to altered neuronal dynamics and impaired neurotransmission in Alpers’ patients. The specific interneuron changes in the white matter indicate impaired neuronal migration, which may suggest neurodevelopmental issues in these patients. Future studies focussing on other brain areas (e.g. frontal lobes) may expand our understanding of Alpers’ neuropathology. M09 Mitochondrial physiology is compromised by mutations in DNAJC3 and may contribute to the clinical presentation of diabetes and neurodegeneration M. Jennings1,2,*, D. Hathazi3,*, M. Synofzik4, A. Roos2,3,*, R. Horvath1,2,* 1 Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; 2The John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle upon Tyne, UK; 3Leibniz-Institut für Analytische Wissenschaften, ISAS, Dortmund, Germany; 4Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany *Equal contributors E-mail: [email protected] Background: Endoplasmic reticulum stress (ER-stress) and dysfunction in the unfolded protein response (UPR), the cellular mechanism resolving ER-stress by up-regulation of protein chaperones and co-chaperones, have been previously associated with both diabetes mellitus and various neurodegenerative diseases. Recently, an inherited chaperonopathy caused by mutations in DNAJC3, which encodes an ER-resident co-chaperone, have been identified in two families presenting diabetes mellitus and widespread degeneration of the central and peripheral nervous system, similar to phenotypes seen in mitochondrial disease. Aims: To elucidate the pathophysiology of DNAJC3-caused phenotype by use of patient-derived fibroblasts using proteomics as an unbiased method to investigate cellular dysfunction, and cell biological approaches to determine how protein alterations affect cellular physiology. Methods: Primary fibroblasts were cultured from DNAJC3 patients and healthy controls, under ER-stress conditions inducted by cholesterol with ACAT (cholesterol metaboliser) inhibitor treatment and under un-stressed conditions. Global proteome profiling was performed to identify proteins affected by loss of functional DNAJC3. Oxygen consumption rate (OCR) measurement (Seahorse) was used to determine the metabolic activity of mitochondria in live fibroblasts. By combining measurement of OCR with injection of drugs inhibiting specific elements of the oxidative phosphorylation system the stress capacity of the mitochondria was determined. Additionally, western blots were performed for DNAJC3 and mitochondrial respiratory complexes. Results: Compared to healthy controls, the proteome profiles of DNAJC3 mutant fibroblasts show alterations in proteins important to mitochondrial physiology. DNAJC3 mutant cell lines exhibit major reductions in oxygen consumption rates corresponding to the maximal rate of oxidative phosphorylation. In contrast, control cell lines show little effect from ER-stress treatment. Conclusions: In this study, a proteomic profile from DNAJC3 mutant fibroblasts indicated alterations in expression affecting mitochondrial function. Further examination of the mitochondrial function in live fibroblasts revealed fundamental disruption of mitochondrial physiology and function when ERstress was induced by cholesterol overload. This provides evidence that dysfunction in the UPR can cause widespread mitochondrial dysfunction associated with cell death, potentially an important mechanism in the pathophysiology of the DNAJC3-chaperonopathy and related conditions.
M10 Expression profiling and bioinformatics analysis of dysregulated microRNAs (miRNAs) in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) M. Levene, B.E. Bax St George’s University of London, Molecular and Cell Sciences Institute, Cell Biology and Genetics Research Centre, London, UK E-mail: [email protected] Background: MNGIE is a rare and fatal inherited metabolic disorder due to a mutation in the nuclear TYMP gene leading to a deficiency in the enzyme thymidine phosphorylase. This subsequently causes an accumulation of the deoxynucleosides, thymidine, and deoxyuridine in tissues and body fluids, imbalances in the mitochondrial deoxyribonucleoside triphosphate pools and ultimately mitochondrial failure. The understanding of the precise molecular mechanisms of this effect and how it influences disease phenotype is poorly understood. MicroRNAs are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level. The aberrant expression of miRNAs is implicated in a wide range of diseases; their study thus offers an approach to the elucidation of the underlying molecular mechanisms of disease pathology. Aims: To examine the expression profile of patient serum and serum derived exosomes with the aim of providing insight into the biochemical derangements which underlie the MNGIE phenotype. Methods: MicroRNA was extracted from patient and healthy control serum and serum-derived exosomes and profiled using Exiqon’s miCURY qPCR panel array. Differentially expressed miRNAs were subjected to bioinformatical analysis. Results: Compared to healthy controls, 53 and 22 up-regulated miRNAs were identified in patient serum and serum-derived exosomes, respectively. Cellular pathway enrichment analysis of the predicted target genes revealed over-representation of 8 pathways in serum, including the T cell receptor, adipocytokine, calcium and MAPK signalling pathways. In exosomes, the Jak-STAT signalling pathway was over-represented. A multiple miRNA target analysis revealed 8 and 6 predicted genes targets in serum and exosomes, respectively that were regulated simultaneously by 5 or more upregulated miRNAs. Conclusions: Expression profiling and bioinformatics analysis of serum-based miRNAs provides a powerful approach for elucidating potential gene targets and target pathways that are affected by deregulated miRNAs M11 Reproductive options in maternally inherited mitochondrial disease: ‘making sense’ J. Maddison1, J. Stewart2, D.M. Turnbull1, T. Rapley3 1 Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; 2 Newcastle upon Tyne NHS Foundation Hospitals, Newcastle upon Tyne, UK; 3 Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK Background: This posters draws on a developing collection of semi-structured interviews (15) with women with maternally inherited mitochondrial disease (MD), offering retrospective and current/prospective accounts of reproductive decision-making. Maternally Inherited Mitochondrial disease is a group of disorders that are heterogeneous and are caused by defects in the mitochondrial DNA (mtDNA). They are a clinically and genetically diverse group of disorders that are progressive and disabling. There are many complexities in mitochondrial biology that make determining and advising on maternally inherited MD particularly problematic compared to other genetic diseases. These bring with them very specific challenges of uncertainty surrounding inheritance risk. With newly emerging reproductive techniques patients have even more reproductive options than ever before to consider. These include pioneering and recently legislated Mitochondrial Donation, Pre-Genetic Diagnosis (PGD), Surrogacy, Ovum Donation, Genetic Counselling, Pre-natal testing, Natural Conception, Adoption and Voluntary Childlessness. Aims: We are interviewing up to 15 women in a retrospective group and up to 15 women who are currently or in the near future thinking about becoming pregnant.
Abstracts of the 10th Neuromuscular Translational Research Conference / Neuromuscular Disorder 27S1 (2017) S5–S44
glutamatergic pyramidal cell populations were quantitatively assessed. The degree of subsequent neuronal loss was also determined. Results: Quantitative quadruple immunofluorescence revealed reduced expression of mitochondrial respiratory chain complexes I and IV within GABAergic interneurons; indicating mitochondrial dysfunction in the parietal tissue of Alpers’ patients. Relative densities of GABAergic interneurons and pyramidal cells were reduced. Mislocalisation of GABAergic interneurons but not pyramidal cells was observed in the white matter of patient parietal tissue. Conclusions: We propose that respiratory deficiencies combined with loss of GABAergic interneurons and pyramidal cells leads to an imbalance between inhibition and excitation, which may contribute to altered neuronal dynamics and impaired neurotransmission in Alpers’ patients. The specific interneuron changes in the white matter indicate impaired neuronal migration, which may suggest neurodevelopmental issues in these patients. Future studies focussing on other brain areas (e.g. frontal lobes) may expand our understanding of Alpers’ neuropathology. M09 Mitochondrial physiology is compromised by mutations in DNAJC3 and may contribute to the clinical presentation of diabetes and neurodegeneration M. Jennings1,2,*, D. Hathazi3,*, M. Synofzik4, A. Roos2,3,*, R. Horvath1,2,* 1 Wellcome Trust Centre for Mitochondrial Research, Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK; 2The John Walton Muscular Dystrophy Research Centre, Institute of Genetic Medicine, Newcastle upon Tyne, UK; 3Leibniz-Institut für Analytische Wissenschaften, ISAS, Dortmund, Germany; 4Department of Neurodegenerative Diseases, Hertie-Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany *Equal contributors E-mail: [email protected] Background: Endoplasmic reticulum stress (ER-stress) and dysfunction in the unfolded protein response (UPR), the cellular mechanism resolving ER-stress by up-regulation of protein chaperones and co-chaperones, have been previously associated with both diabetes mellitus and various neurodegenerative diseases. Recently, an inherited chaperonopathy caused by mutations in DNAJC3, which encodes an ER-resident co-chaperone, have been identified in two families presenting diabetes mellitus and widespread degeneration of the central and peripheral nervous system, similar to phenotypes seen in mitochondrial disease. Aims: To elucidate the pathophysiology of DNAJC3-caused phenotype by use of patient-derived fibroblasts using proteomics as an unbiased method to investigate cellular dysfunction, and cell biological approaches to determine how protein alterations affect cellular physiology. Methods: Primary fibroblasts were cultured from DNAJC3 patients and healthy controls, under ER-stress conditions inducted by cholesterol with ACAT (cholesterol metaboliser) inhibitor treatment and under un-stressed conditions. Global proteome profiling was performed to identify proteins affected by loss of functional DNAJC3. Oxygen consumption rate (OCR) measurement (Seahorse) was used to determine the metabolic activity of mitochondria in live fibroblasts. By combining measurement of OCR with injection of drugs inhibiting specific elements of the oxidative phosphorylation system the stress capacity of the mitochondria was determined. Additionally, western blots were performed for DNAJC3 and mitochondrial respiratory complexes. Results: Compared to healthy controls, the proteome profiles of DNAJC3 mutant fibroblasts show alterations in proteins important to mitochondrial physiology. DNAJC3 mutant cell lines exhibit major reductions in oxygen consumption rates corresponding to the maximal rate of oxidative phosphorylation. In contrast, control cell lines show little effect from ER-stress treatment. Conclusions: In this study, a proteomic profile from DNAJC3 mutant fibroblasts indicated alterations in expression affecting mitochondrial function. Further examination of the mitochondrial function in live fibroblasts revealed fundamental disruption of mitochondrial physiology and function when ERstress was induced by cholesterol overload. This provides evidence that dysfunction in the UPR can cause widespread mitochondrial dysfunction associated with cell death, potentially an important mechanism in the pathophysiology of the DNAJC3-chaperonopathy and related conditions.
M10 Expression profiling and bioinformatics analysis of dysregulated microRNAs (miRNAs) in mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) M. Levene, B.E. Bax St George’s University of London, Molecular and Cell Sciences Institute, Cell Biology and Genetics Research Centre, London, UK E-mail: [email protected] Background: MNGIE is a rare and fatal inherited metabolic disorder due to a mutation in the nuclear TYMP gene leading to a deficiency in the enzyme thymidine phosphorylase. This subsequently causes an accumulation of the deoxynucleosides, thymidine, and deoxyuridine in tissues and body fluids, imbalances in the mitochondrial deoxyribonucleoside triphosphate pools and ultimately mitochondrial failure. The understanding of the precise molecular mechanisms of this effect and how it influences disease phenotype is poorly understood. MicroRNAs are a class of small non-coding RNAs that regulate gene expression at the posttranscriptional level. The aberrant expression of miRNAs is implicated in a wide range of diseases; their study thus offers an approach to the elucidation of the underlying molecular mechanisms of disease pathology. Aims: To examine the expression profile of patient serum and serum derived exosomes with the aim of providing insight into the biochemical derangements which underlie the MNGIE phenotype. Methods: MicroRNA was extracted from patient and healthy control serum and serum-derived exosomes and profiled using Exiqon’s miCURY qPCR panel array. Differentially expressed miRNAs were subjected to bioinformatical analysis. Results: Compared to healthy controls, 53 and 22 up-regulated miRNAs were identified in patient serum and serum-derived exosomes, respectively. Cellular pathway enrichment analysis of the predicted target genes revealed over-representation of 8 pathways in serum, including the T cell receptor, adipocytokine, calcium and MAPK signalling pathways. In exosomes, the Jak-STAT signalling pathway was over-represented. A multiple miRNA target analysis revealed 8 and 6 predicted genes targets in serum and exosomes, respectively that were regulated simultaneously by 5 or more upregulated miRNAs. Conclusions: Expression profiling and bioinformatics analysis of serum-based miRNAs provides a powerful approach for elucidating potential gene targets and target pathways that are affected by deregulated miRNAs M11 Reproductive options in maternally inherited mitochondrial disease: ‘making sense’ J. Maddison1, J. Stewart2, D.M. Turnbull1, T. Rapley3 1 Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK; 2 Newcastle upon Tyne NHS Foundation Hospitals, Newcastle upon Tyne, UK; 3 Institute of Health and Society, Newcastle University, Newcastle upon Tyne, UK Background: This posters draws on a developing collection of semi-structured interviews (15) with women with maternally inherited mitochondrial disease (MD), offering retrospective and current/prospective accounts of reproductive decision-making. Maternally Inherited Mitochondrial disease is a group of disorders that are heterogeneous and are caused by defects in the mitochondrial DNA (mtDNA). They are a clinically and genetically diverse group of disorders that are progressive and disabling. There are many complexities in mitochondrial biology that make determining and advising on maternally inherited MD particularly problematic compared to other genetic diseases. These bring with them very specific challenges of uncertainty surrounding inheritance risk. With newly emerging reproductive techniques patients have even more reproductive options than ever before to consider. These include pioneering and recently legislated Mitochondrial Donation, Pre-Genetic Diagnosis (PGD), Surrogacy, Ovum Donation, Genetic Counselling, Pre-natal testing, Natural Conception, Adoption and Voluntary Childlessness. Aims: We are interviewing up to 15 women in a retrospective group and up to 15 women who are currently or in the near future thinking about becoming pregnant.