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Children with mitochondrial disease and autism have alterations in pathways involved in response to endogenous and exogenous stressors
558
Abstracts
or Tempol‐H did not restore cell survival. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF24 analyzer. As seen previously, AZT/ddI reduced maximal FCCP-uncoupled OCR. However co-exposure with Tempol and Tempol-H stimulated OCR, restoring the AZT/ddIreduced uncoupled OCR by 22–91%. Similarly, the uncoupled ECAR levels were increased by 15–46% with Tempol or Tempol-H. Preliminary Western blot findings showed that Tempol and Tempol-H enhanced the expression of uncoupling protein-2 (UCP-2). Therefore Tempol and Tempol-H may protect cardiomyocytes from mitochondrial compromise induced by the NRTI combination AZT/ddI, and UCP-2 may play a role through mild uncoupling. We are currently evaluating potential mechanisms by which these compounds may act as mitochondrial protective agents. doi:10.1016/j.mito.2012.07.021
23 Children with mitochondrial disease and autism have alterations in pathways involved in response to endogenous and exogenous stressors Presenter: Richard E. Frye Richard E. Frye, Shannon Rose, S. Jill James Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA 72202 Background: Mitochondrial disease (MD) is an important medical co-morbidity associated with autism spectrum disorder (ASD). Children with ASD and MD (ASD/MD) make up an important subgroup of children with ASD but little is known about their physiological, developmental and genetic characteristics as compared to children with ASD but no MD (ASD/NoMD). In addition, the majority of ASD/MD cases cannot be explained by a known mitochondrial DNA abnormality leaving open the possibility that their MD could be secondary to other physiological abnormalities, such as oxidative stress, or associated with nuclear genetic lesions. Methods: 18 ASD/MD children were compared to 18 ASD/NoMD children matched on age and gender. Measures of language development, adaptive behavior, core autism symptoms, oxidative stress [plasma glutathione, methylation precursors and 3-nitrotyrosine (3NT)], inflammation [3-chlorotyrosine (3-CT)], genome-wide gene expression and candidate nucleotide polymorphisms were obtained. Expression of nuclear electron transport chain (ETC) complex genes was also examined. Enrichment analysis was conducted to match significant genetic differences in expression and polymorphisms between the groups to functional ontologies derived from canonical pathways. Results: The ASD/MD group demonstrated higher 3-CT as compared to ASD/NoMD suggesting greater immune activation and inflammation in the ASD/MD group. The relationship between 3-NT, a measure of chronic oxidative stress, and adaptive behavior, language development and core autism symptoms was different for the two groups. Better adaptive behavior, language development and fewer core autism symptoms were associated with higher 3-NT levels in the ASD/MD group but lower 3-NT levels in the ASD/NoMD group. Similar, but fewer, relationships were also found for 3-CT. The relationship between the expression of ETC nuclear genes was also differentially related to 3-NT and 3-CT between the two groups. Enrichment analysis found that both expression and polymorphism differences strongly converged on the pathway responsible for maturation of pro-opiomelanocortin, a pathway that is essential for the production of adrenocorticotropic hormone. Conclusions: Clear physiological differences were found between the ASD children with and without MD. Both chronic oxidative stress and inflammation appear to be differentially related to measures of
cognitive development and gene expression between the two groups. Lastly, children with ASD/MD appear to have genetic polymorphisms that effect gene expression related to hormonal pathways responsible for adaptation to physiological stressors. Together, these data suggest that children with ASD/MD have a unique profile of pathways involved in adapting to endogenous and exogenous stressors. These findings have significant implications for treatment of this subgroup of ASD children.
doi:10.1016/j.mito.2012.07.022
24 A mouse model with a missense mutation in ND6 for pre-Leber's hereditary optic neuropathy Presenter: Chun Shi Lin Chun Shi Lina,b, Mark Sharpleya,b, Megan McManusb, Eric Sungc, Billy Phand, Fred Ross-Cisnerosd, Peter Baciuc, Valerio Carellie, Alfredo Sadund, Douglas Wallacea,b a Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, United States b Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States c Allergan Inc., Irvine, CA 92623, United States d Doheny Eye Institute, Department of Ophthalmology, USC-Keck School of Medicine, Los Angeles, CA 90089, United States e Department of Neurological Sciences, University of Bologna, Via Ugo Foscolo 7, 40123 Bologna, Italy Mitochondrial DNA (mtDNA) mutations have long been implicated in ophthalmological and neuronal disorders. More specifically, mtDNA mutations in the subunits of NADH: ubiquinone oxidoreductase (complex I) cause Leber's hereditary optic neuropathy (LHON). Roughly 95% of patients harbor one of three point mutations in mtDNA encoding complex I subunit: G3460A of ND1, G11778A of ND4, and T14484C of ND6. LHON patients suffer acute vision losses due to massive retinal ganglion cell losses. Visual recovery is rare, and treatment is scarce. Clinical and pathological studies for LHON are limited to biochemical measurements of in vitro cybrids and pathology on post-mortem samples. Cybrids are immortalized cell lines that do not share physiological relevance and complexity of LHON patients. Histopathology does not address the progression, rather the end point, of the disease. As a result, attempts to understand the consequences of LHON mutations have yielded limited and conflicting interpretations. To adequately remedy this problem, a homoplasmic missense, G13997A, mtDNA mutation, has been introduced into the mouse germline. This mutation results in an amino acid change of P25L in the ND6 subunit of complex I. The ND6 mutant mice displayed many of the same characteristics of asymptomatic LHON mutation carriers and patients. While the vision was not impaired, the retinal response of the mutant mice was significantly decreased under light and dark conditions. Pathology on the optic nerve of ND6 mutant mice revealed retinal cell death and swollen axonal population. In addition, ND6 axons possessed proliferative, yet abnormal mitochondria. Biochemical measurements determined that the ND6 mutant suffered from reduced bioenergetic capacity driven by complex I and increased oxidative stress and injuries. This transgenic strain is the first mouse model that genetically, phenotypically, and biochemically recapitulates all aspects of LHON. It provides an opportunity to both test the efficacy of therapies and determine which factors modulate the penetrance of LHON.
doi:10.1016/j.mito.2012.07.023
Abstracts
or Tempol‐H did not restore cell survival. Mitochondrial oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF24 analyzer. As seen previously, AZT/ddI reduced maximal FCCP-uncoupled OCR. However co-exposure with Tempol and Tempol-H stimulated OCR, restoring the AZT/ddIreduced uncoupled OCR by 22–91%. Similarly, the uncoupled ECAR levels were increased by 15–46% with Tempol or Tempol-H. Preliminary Western blot findings showed that Tempol and Tempol-H enhanced the expression of uncoupling protein-2 (UCP-2). Therefore Tempol and Tempol-H may protect cardiomyocytes from mitochondrial compromise induced by the NRTI combination AZT/ddI, and UCP-2 may play a role through mild uncoupling. We are currently evaluating potential mechanisms by which these compounds may act as mitochondrial protective agents. doi:10.1016/j.mito.2012.07.021
23 Children with mitochondrial disease and autism have alterations in pathways involved in response to endogenous and exogenous stressors Presenter: Richard E. Frye Richard E. Frye, Shannon Rose, S. Jill James Arkansas Children's Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA 72202 Background: Mitochondrial disease (MD) is an important medical co-morbidity associated with autism spectrum disorder (ASD). Children with ASD and MD (ASD/MD) make up an important subgroup of children with ASD but little is known about their physiological, developmental and genetic characteristics as compared to children with ASD but no MD (ASD/NoMD). In addition, the majority of ASD/MD cases cannot be explained by a known mitochondrial DNA abnormality leaving open the possibility that their MD could be secondary to other physiological abnormalities, such as oxidative stress, or associated with nuclear genetic lesions. Methods: 18 ASD/MD children were compared to 18 ASD/NoMD children matched on age and gender. Measures of language development, adaptive behavior, core autism symptoms, oxidative stress [plasma glutathione, methylation precursors and 3-nitrotyrosine (3NT)], inflammation [3-chlorotyrosine (3-CT)], genome-wide gene expression and candidate nucleotide polymorphisms were obtained. Expression of nuclear electron transport chain (ETC) complex genes was also examined. Enrichment analysis was conducted to match significant genetic differences in expression and polymorphisms between the groups to functional ontologies derived from canonical pathways. Results: The ASD/MD group demonstrated higher 3-CT as compared to ASD/NoMD suggesting greater immune activation and inflammation in the ASD/MD group. The relationship between 3-NT, a measure of chronic oxidative stress, and adaptive behavior, language development and core autism symptoms was different for the two groups. Better adaptive behavior, language development and fewer core autism symptoms were associated with higher 3-NT levels in the ASD/MD group but lower 3-NT levels in the ASD/NoMD group. Similar, but fewer, relationships were also found for 3-CT. The relationship between the expression of ETC nuclear genes was also differentially related to 3-NT and 3-CT between the two groups. Enrichment analysis found that both expression and polymorphism differences strongly converged on the pathway responsible for maturation of pro-opiomelanocortin, a pathway that is essential for the production of adrenocorticotropic hormone. Conclusions: Clear physiological differences were found between the ASD children with and without MD. Both chronic oxidative stress and inflammation appear to be differentially related to measures of
cognitive development and gene expression between the two groups. Lastly, children with ASD/MD appear to have genetic polymorphisms that effect gene expression related to hormonal pathways responsible for adaptation to physiological stressors. Together, these data suggest that children with ASD/MD have a unique profile of pathways involved in adapting to endogenous and exogenous stressors. These findings have significant implications for treatment of this subgroup of ASD children.
doi:10.1016/j.mito.2012.07.022
24 A mouse model with a missense mutation in ND6 for pre-Leber's hereditary optic neuropathy Presenter: Chun Shi Lin Chun Shi Lina,b, Mark Sharpleya,b, Megan McManusb, Eric Sungc, Billy Phand, Fred Ross-Cisnerosd, Peter Baciuc, Valerio Carellie, Alfredo Sadund, Douglas Wallacea,b a Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, United States b Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia, Philadelphia, PA 19104, United States c Allergan Inc., Irvine, CA 92623, United States d Doheny Eye Institute, Department of Ophthalmology, USC-Keck School of Medicine, Los Angeles, CA 90089, United States e Department of Neurological Sciences, University of Bologna, Via Ugo Foscolo 7, 40123 Bologna, Italy Mitochondrial DNA (mtDNA) mutations have long been implicated in ophthalmological and neuronal disorders. More specifically, mtDNA mutations in the subunits of NADH: ubiquinone oxidoreductase (complex I) cause Leber's hereditary optic neuropathy (LHON). Roughly 95% of patients harbor one of three point mutations in mtDNA encoding complex I subunit: G3460A of ND1, G11778A of ND4, and T14484C of ND6. LHON patients suffer acute vision losses due to massive retinal ganglion cell losses. Visual recovery is rare, and treatment is scarce. Clinical and pathological studies for LHON are limited to biochemical measurements of in vitro cybrids and pathology on post-mortem samples. Cybrids are immortalized cell lines that do not share physiological relevance and complexity of LHON patients. Histopathology does not address the progression, rather the end point, of the disease. As a result, attempts to understand the consequences of LHON mutations have yielded limited and conflicting interpretations. To adequately remedy this problem, a homoplasmic missense, G13997A, mtDNA mutation, has been introduced into the mouse germline. This mutation results in an amino acid change of P25L in the ND6 subunit of complex I. The ND6 mutant mice displayed many of the same characteristics of asymptomatic LHON mutation carriers and patients. While the vision was not impaired, the retinal response of the mutant mice was significantly decreased under light and dark conditions. Pathology on the optic nerve of ND6 mutant mice revealed retinal cell death and swollen axonal population. In addition, ND6 axons possessed proliferative, yet abnormal mitochondria. Biochemical measurements determined that the ND6 mutant suffered from reduced bioenergetic capacity driven by complex I and increased oxidative stress and injuries. This transgenic strain is the first mouse model that genetically, phenotypically, and biochemically recapitulates all aspects of LHON. It provides an opportunity to both test the efficacy of therapies and determine which factors modulate the penetrance of LHON.
doi:10.1016/j.mito.2012.07.023