- Email: [email protected]
Chronic pain, fatigue, GI dysmotility and the mitochondrial chaperone TRAP1
S24
Abstracts
MNRR1. Cells with increased levels of MNRR1 show increased oxygen consumption and cells with reduced levels of MNRR1 (KD cells) show reduced oxygen consumption; KD cells also show increased ROS production, reduced growth rate and fragmented mitochondria. MNRR1 requires phosphorylation at Tyr-99, carried out in vivo by Abl kinase, for efficient binding to COX; cells containing the Y99F replacement show the phenotype of KD cells. A related CX9C protein, CHCHD10, is also present in both the mitochondria and the nucleus. In the mitochondria it also binds to COX and also causes increased oxygen consumption; furthermore, it interacts with MNRR1. A family with a severe form of Charcot-Marie-Tooth (CMT) disease type 1A, caused by a duplication of PMP22, also carries a Q112H mutation in MNRR1. This mutation reduces the ability of Abl kinase to bind to MNRR1 and to phosphorylate it at Y99, and it reduces the ability of MNRR1 to bind to COX and to promote transcription of a COX4I2 reporter. This mutation thus represents a modifier gene whereby a reduction of mitochondrial function can exacerbate the phenotype of a non-mitochondrial disease mutation. doi:10.1016/j.mito.2015.07.069
Abstract 57 Chronic pain, fatigue, GI dysmotility and the mitochondrial chaperone TRAP1 Presenter: Richard G. Boles Richard G. Bolesa, Holly A. Hornunga, Alastair Moodya, Thomas Ortiza, Stacey A. Wonga, Julie M. Eggingtona, Christine M. Stanleya, Mu Gaob, Hongyi Zhoub, Amir S. Zarea, Jeffrey Skolnickb, Kevin J. McKernana a Courtagen Life Sciences, Woburn, MA, USA b Center for the Study of Systems Biology, Georgia Institute of Technology, Atlanta, GA, USA Body of Abstract: “Functional” disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. Functional symptomatology such as chronic pain, fatigue and nausea are some of the most common complaints of patients with a variety of mitochondrial disorders, and increased oxidative damage has been reported in patients with some functional disorders. Thus, an excellent candidate gene in functional symptom pathogenesis is TRAP1, which encodes a mitochondrial chaperone (AKA: HSP90) that plays a role in protecting mitochondria by reducing reactive oxygen species (ROS) levels. In this study, clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients referred to Courtagen for clinical diagnostics for a suspected mitochondrial disorder. Among the 930 patients, the combined presence of the three functional categories (“triad”) is strongly associated with very-highly evolutionarily conserved TRAP1 variants within the ATPase binding pocket (amino acids 108-260), but not in such conserved variants in other domains of the 701amino acid TRAP1 protein (OR = 12, 95% confidence interval = 3.049, P = 0.0005). In particular, compared against our patients without any TRAP1 variants, the functional triad is strongly associated with p.Ile253Val (OR = 7.5, 95%CI = 2.8-20, P = 0.0001), which may define the depth of the ATP binding pocket, and with two other variants, p.Arg128His and p.Glu192Lys (OR = 18, 95%CI = 3.6-100, P = 0.0005), which our computer model suggests form a salt bridge to close the ATP binding pocket's “lid”. Additionally, these three variants together are associated with the individual symptoms of chronic pain, chronic fatigue and GI dysmotility (odds ratios = 3.65.6, P = 0.0002-0.005). Anecdotal experience suggests that highly aggressive antioxidant therapy may have substantial clinic utility in patients with these variants. Considering variant population prevalence rates of 0.61%, 0.32%, and 0.14% by ExAC, respectively, and high
odds ratios, these variants may be an important factor in the etiology of multiple functional symptomatology, likely acting as a permissive genetic factor in a multifactorial model. doi:10.1016/j.mito.2015.07.070
Abstract 58 AAV2-mediated delivery of nucleic acid or protein to the mitochondrial matrix as a treatment for Lebers Hereditary Optic Neuropathy Presenter: Marshall W. Huston Marshall W. Hustona, Pier G. Mastroberardinob, Jeroen de Vrija, Bert H. Smeetsc, Gerard Wagemakerd, René I. deCooa a Department of Neurology, Erasmus University Medical Center, Rotterdam, the Netherlands b Department of Genetics, Erasmus University Medical Center, the Netherlands c Department of Genetics and Cell Biology, Maastricht University Medical Center, Maastricht, the Netherlands d Hacettepe University, Ankara, Turkey Body of Abstract: Lebers Hereditary Optic Neuropathy (LHON) is form of inherited blindness caused by point mutations in Complex I subunit genes ND1, ND4 or ND6, with the ND4 11778GNA mutation being most common. Due to the presence of these genes in the mitochondrial chromosome, conventional gene therapy tools specialized for delivering therapeutic genes to the nucleus must rely on allotopic expression and cytosolic transport of the ND proteins. This process can be effective but is complicated by the highly hydrophobic nature of these proteins. To facilitate transcription and translation of the ND genes in their natural environment, i.e. the mitochondrial matrix, an AAV2 vector was modified by introducing a mitochondrial targeting sequence (MTS) into the reading frame of capsid protein VP2, creating an mito-targeted vector. We recoded GFP for mitochondrial-exclusive translation (mitoGFP) for an unambiguous visual readout to identify cells where vector-delivered transgene expression was occurring in mitochondria. LHON cybrid cells containing the 11778GNA mutation in 100% of their mitochondria were created from LHON patient fibroblasts. Complex I activity in these cybrids was measured via Seahorse analysis and found to be deficient compared to cells containing wild type mitochondria. Future experiments will compare the efficacy of the mito-targeted vectors containing wild type ND4 to allotopic vectors containing nuclear-encoded ND4 to determine which is more effective at improving Complex I activity and survival of LHON cybrid cells, as well as preventing or reversing vision loss in a LHON animal model, at a clinically relevant viral titer. doi:10.1016/j.mito.2015.07.071
Abstract 59 Cyclophilin-D: A Novel regulator of mitochondrial gene expression Presenter: Raúl J. Gazmuri MD Jeejabai Radhakrishnan, Raúl J. Gazmuri Department of Medicine, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Resuscitation Institute at Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Captain James A. Lovell Federal Health Care Center, North Chicago, IL, USA Abstract: Cyclophilin-D (Cyp-D) is a peptidyl prolyl isomerase that catalyzes the cis-trans isomerization of peptidyl prolyl bonds and therefore regulates conformational changes of target proteins.
Abstracts
MNRR1. Cells with increased levels of MNRR1 show increased oxygen consumption and cells with reduced levels of MNRR1 (KD cells) show reduced oxygen consumption; KD cells also show increased ROS production, reduced growth rate and fragmented mitochondria. MNRR1 requires phosphorylation at Tyr-99, carried out in vivo by Abl kinase, for efficient binding to COX; cells containing the Y99F replacement show the phenotype of KD cells. A related CX9C protein, CHCHD10, is also present in both the mitochondria and the nucleus. In the mitochondria it also binds to COX and also causes increased oxygen consumption; furthermore, it interacts with MNRR1. A family with a severe form of Charcot-Marie-Tooth (CMT) disease type 1A, caused by a duplication of PMP22, also carries a Q112H mutation in MNRR1. This mutation reduces the ability of Abl kinase to bind to MNRR1 and to phosphorylate it at Y99, and it reduces the ability of MNRR1 to bind to COX and to promote transcription of a COX4I2 reporter. This mutation thus represents a modifier gene whereby a reduction of mitochondrial function can exacerbate the phenotype of a non-mitochondrial disease mutation. doi:10.1016/j.mito.2015.07.069
Abstract 57 Chronic pain, fatigue, GI dysmotility and the mitochondrial chaperone TRAP1 Presenter: Richard G. Boles Richard G. Bolesa, Holly A. Hornunga, Alastair Moodya, Thomas Ortiza, Stacey A. Wonga, Julie M. Eggingtona, Christine M. Stanleya, Mu Gaob, Hongyi Zhoub, Amir S. Zarea, Jeffrey Skolnickb, Kevin J. McKernana a Courtagen Life Sciences, Woburn, MA, USA b Center for the Study of Systems Biology, Georgia Institute of Technology, Atlanta, GA, USA Body of Abstract: “Functional” disorders are common conditions with a substantial impact on a patients' wellbeing, and can be diagnostically elusive. Functional symptomatology such as chronic pain, fatigue and nausea are some of the most common complaints of patients with a variety of mitochondrial disorders, and increased oxidative damage has been reported in patients with some functional disorders. Thus, an excellent candidate gene in functional symptom pathogenesis is TRAP1, which encodes a mitochondrial chaperone (AKA: HSP90) that plays a role in protecting mitochondria by reducing reactive oxygen species (ROS) levels. In this study, clinical information and the exon sequence of the TRAP1 mitochondrial chaperone were reviewed with a focus on the functional categories of chronic pain, fatigue and gastrointestinal dysmotility. Very-highly conserved TRAP1 variants were identified in 73 of 930 unrelated patients referred to Courtagen for clinical diagnostics for a suspected mitochondrial disorder. Among the 930 patients, the combined presence of the three functional categories (“triad”) is strongly associated with very-highly evolutionarily conserved TRAP1 variants within the ATPase binding pocket (amino acids 108-260), but not in such conserved variants in other domains of the 701amino acid TRAP1 protein (OR = 12, 95% confidence interval = 3.049, P = 0.0005). In particular, compared against our patients without any TRAP1 variants, the functional triad is strongly associated with p.Ile253Val (OR = 7.5, 95%CI = 2.8-20, P = 0.0001), which may define the depth of the ATP binding pocket, and with two other variants, p.Arg128His and p.Glu192Lys (OR = 18, 95%CI = 3.6-100, P = 0.0005), which our computer model suggests form a salt bridge to close the ATP binding pocket's “lid”. Additionally, these three variants together are associated with the individual symptoms of chronic pain, chronic fatigue and GI dysmotility (odds ratios = 3.65.6, P = 0.0002-0.005). Anecdotal experience suggests that highly aggressive antioxidant therapy may have substantial clinic utility in patients with these variants. Considering variant population prevalence rates of 0.61%, 0.32%, and 0.14% by ExAC, respectively, and high
odds ratios, these variants may be an important factor in the etiology of multiple functional symptomatology, likely acting as a permissive genetic factor in a multifactorial model. doi:10.1016/j.mito.2015.07.070
Abstract 58 AAV2-mediated delivery of nucleic acid or protein to the mitochondrial matrix as a treatment for Lebers Hereditary Optic Neuropathy Presenter: Marshall W. Huston Marshall W. Hustona, Pier G. Mastroberardinob, Jeroen de Vrija, Bert H. Smeetsc, Gerard Wagemakerd, René I. deCooa a Department of Neurology, Erasmus University Medical Center, Rotterdam, the Netherlands b Department of Genetics, Erasmus University Medical Center, the Netherlands c Department of Genetics and Cell Biology, Maastricht University Medical Center, Maastricht, the Netherlands d Hacettepe University, Ankara, Turkey Body of Abstract: Lebers Hereditary Optic Neuropathy (LHON) is form of inherited blindness caused by point mutations in Complex I subunit genes ND1, ND4 or ND6, with the ND4 11778GNA mutation being most common. Due to the presence of these genes in the mitochondrial chromosome, conventional gene therapy tools specialized for delivering therapeutic genes to the nucleus must rely on allotopic expression and cytosolic transport of the ND proteins. This process can be effective but is complicated by the highly hydrophobic nature of these proteins. To facilitate transcription and translation of the ND genes in their natural environment, i.e. the mitochondrial matrix, an AAV2 vector was modified by introducing a mitochondrial targeting sequence (MTS) into the reading frame of capsid protein VP2, creating an mito-targeted vector. We recoded GFP for mitochondrial-exclusive translation (mitoGFP) for an unambiguous visual readout to identify cells where vector-delivered transgene expression was occurring in mitochondria. LHON cybrid cells containing the 11778GNA mutation in 100% of their mitochondria were created from LHON patient fibroblasts. Complex I activity in these cybrids was measured via Seahorse analysis and found to be deficient compared to cells containing wild type mitochondria. Future experiments will compare the efficacy of the mito-targeted vectors containing wild type ND4 to allotopic vectors containing nuclear-encoded ND4 to determine which is more effective at improving Complex I activity and survival of LHON cybrid cells, as well as preventing or reversing vision loss in a LHON animal model, at a clinically relevant viral titer. doi:10.1016/j.mito.2015.07.071
Abstract 59 Cyclophilin-D: A Novel regulator of mitochondrial gene expression Presenter: Raúl J. Gazmuri MD Jeejabai Radhakrishnan, Raúl J. Gazmuri Department of Medicine, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Resuscitation Institute at Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA Captain James A. Lovell Federal Health Care Center, North Chicago, IL, USA Abstract: Cyclophilin-D (Cyp-D) is a peptidyl prolyl isomerase that catalyzes the cis-trans isomerization of peptidyl prolyl bonds and therefore regulates conformational changes of target proteins.