RESUMO
Autosomal dominant optic atrophy (DOA) is a progressive form of blindness caused by degeneration of retinal ganglion cells and their axons, mainly caused by mutations in the OPA1 mitochondrial dynamin like GTPase (OPA1) gene. OPA1 encodes a dynamin-like GTPase present in the mitochondrial inner membrane. When associated with OPA1 mutations, DOA can present not only ocular symptoms but also multi-organ symptoms (DOA plus). DOA plus often results from point mutations in the GTPase domain, which are assumed to have dominant-negative effects. However, the presence of mutations in the GTPase domain does not always result in DOA plus. Therefore, an experimental system to distinguish between DOA and DOA plus is needed. In this study, we found that loss-of-function mutations of the dOPA1 gene in Drosophila can imitate the pathology of optic nerve degeneration observed in DOA. We successfully rescued this degeneration by expressing the human OPA1 (hOPA1) gene, indicating that hOPA1 is functionally interchangeable with dOPA1 in the fly system. However, mutations previously identified did not ameliorate the dOPA1 deficiency phenotype. By expressing both WT and DOA plus mutant hOPA1 forms in the optic nerve of dOPA1 mutants, we observed that DOA plus mutations suppressed the rescue, facilitating the distinction between loss-of-function and dominant-negative mutations in hOPA1. This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies.
Assuntos
Modelos Animais de Doenças , Proteínas de Drosophila , GTP Fosfo-Hidrolases , Atrofia Óptica Autossômica Dominante , Animais , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Humanos , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Mutação , Drosophila/genética , Proteínas de MembranaRESUMO
Autosomal dominant optic atrophy (ADOA) is a rare progressive disease mainly caused by mutations in OPA1, a nuclear gene encoding for a mitochondrial protein that plays an essential role in mitochondrial dynamics, cell survival, oxidative phosphorylation, and mtDNA maintenance. ADOA is characterized by the degeneration of retinal ganglion cells (RGCs). This causes visual loss, which can lead to legal blindness in many cases. Nowadays, there is no effective treatment for ADOA. In this article, we have established an isogenic human RGC model for ADOA using iPSC technology and the genome editing tool CRISPR/Cas9 from a previously generated iPSC line of an ADOA plus patient harboring the pathogenic variant NM_015560.3: c.1861C>T (p.Gln621Ter) in heterozygosis in OPA1. To this end, a protocol based on supplementing the iPSC culture media with several small molecules and defined factors trying to mimic embryonic development has been employed. Subsequently, the created model was validated, confirming the presence of a defect of intergenomic communication, impaired mitochondrial respiration, and an increase in apoptosis and ROS generation. Finally, we propose the analysis of OPA1 expression by qPCR as an easy read-out method to carry out future drug screening studies using the created RGC model. In summary, this model provides a useful platform for further investigation of the underlying pathophysiological mechanisms of ADOA plus and for testing compounds with potential pharmacological action.
Assuntos
GTP Fosfo-Hidrolases , Células-Tronco Pluripotentes Induzidas , Atrofia Óptica Autossômica Dominante , Células Ganglionares da Retina , Humanos , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/patologia , Atrofia Óptica Autossômica Dominante/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Sistemas CRISPR-Cas , Edição de Genes/métodos , Mutação , Apoptose/genética , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/genéticaRESUMO
Autosomal dominant optic atrophy plus (ADOA+) is characterized by primary optic nerve atrophy accompanied by a spectrum of degenerative neurological symptoms. Despite ongoing research, no effective treatments are currently available for this condition. Our study provided evidence for the pathogenicity of an unreported c.1780T>C variant in the OPA1 gene through patient-derived skin fibroblasts and an engineered HEK293T cell line with OPA1 downregulation. We demonstrate that OPA1 insufficiency promoted mitochondrial fragmentation and increased DRP1 expression, disrupting mitochondrial dynamics. Consequently, this disruption enhanced mitophagy and caused mitochondrial dysfunction, contributing to the ADOA+ phenotype. Notably, the Drp1 inhibitor, mitochondrial division inhibitor-1 (Mdivi-1), effectively mitigated the adverse effects of OPA1 impairment. These effects included reduced Drp1 phosphorylation, decreased mitochondrial fragmentation, and balanced mitophagy. Thus, we propose that intervening in DRP1 with Mdivi-1 could correct mitochondrial abnormalities, offering a promising therapeutic approach for managing ADOA+.
Assuntos
Dinaminas , Fibroblastos , GTP Fosfo-Hidrolases , Mitocôndrias , Mitofagia , Atrofia Óptica Autossômica Dominante , Quinazolinonas , Humanos , Dinaminas/genética , Dinaminas/metabolismo , Dinaminas/antagonistas & inibidores , Quinazolinonas/farmacologia , Quinazolinonas/uso terapêutico , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/patologia , Atrofia Óptica Autossômica Dominante/tratamento farmacológico , Atrofia Óptica Autossômica Dominante/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mitofagia/efeitos dos fármacos , Células HEK293 , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Masculino , FemininoRESUMO
Mitochondrial dynamics homeostasis is sustained by continuous and balanced fission and fusion, which are determinants of morphology, abundance, biogenesis and mitophagy of mitochondria. Optic atrophy 1 (OPA1), as the only inner mitochondrial membrane fusion protein, plays a key role in stabilizing mitochondrial dynamics. The disturbance of mitochondrial dynamics contributes to the pathophysiological progress of cardiovascular disorders, which are the main cause of death worldwide in recent decades and result in tremendous social burden. In this review, we describe the latest findings regarding OPA1 and its role in mitochondrial fusion. We summarize the post-translational modifications (PTMs) for OPA1 and its regulatory role in mitochondrial dynamics. Then the diverse cell fates caused by OPA1 expression during cardiovascular disorders are discussed. Moreover, cardiovascular disorders (such as heart failure, myocardial ischemia/reperfusion injury, cardiomyopathy and cardiac hypertrophy) relevant to OPA1-dependent mitochondrial dynamics imbalance have been detailed. Finally, we highlight the potential that targeting OPA1 to impact mitochondrial fusion may be used as a novel strategy against cardiovascular disorders.
Assuntos
Cardiomiopatias , Insuficiência Cardíaca , Atrofia Óptica Autossômica Dominante , Humanos , Dinâmica Mitocondrial , Atrofia Óptica Autossômica Dominante/metabolismo , Cardiomiopatias/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
Autosomal dominant optic atrophy (ADOA), mostly caused by heterozygous OPA1 mutations and characterized by retinal ganglion cell (RGC) loss and optic nerve degeneration, is one of the most common types of inherited optic neuropathies. Previous work using a two-dimensional (2D) differentiation model of induced pluripotent stem cells (iPSCs) has investigated ADOA pathogenesis but failed to agree on the effect of OPA1 mutations on RGC differentiation. Here, we use 3D retinal organoids capable of mimicking in vivo retinal development to resolve the issue. We generated isogenic iPSCs carrying the hotspot OPA1 c.2708_2711delTTAG mutation and found that the mutant variant caused defective initial and terminal differentiation and abnormal electrophysiological properties of organoid-derived RGCs. Moreover, this variant inhibits progenitor proliferation and results in mitochondrial dysfunction. These data demonstrate that retinal organoids coupled with gene editing serve as a powerful tool to definitively identify disease-related phenotypes and provide valuable resources to further investigate ADOA pathogenesis and screen for ADOA therapeutics.
Assuntos
Atrofia Óptica Autossômica Dominante , Células Ganglionares da Retina , Humanos , Células Ganglionares da Retina/metabolismo , Retina/metabolismo , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Mutação , Diferenciação Celular/genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismoRESUMO
Mitochondria are integrative hubs central to cellular adaptive pathways. Such pathways are critical in highly differentiated postmitotic neurons, the plasticity of which sustains brain function. Consequently, defects in mitochondria and in their dynamics appear instrumental in neurodegenerative diseases and may also participate in cognitive impairments. To directly test this hypothesis, we analyzed cognitive performances in a mouse mitochondria-based disease model, because of haploinsufficiency in the mitochondrial optic atrophy type 1 (OPA1) protein involved in mitochondrial dynamics. In males, we evaluated adult hippocampal neurogenesis parameters using immunohistochemistry. We performed a battery of tests to assess basal behavioral characteristics and cognitive performances, and tested putative treatments. While in dominant optic atrophy (DOA) mouse models, the known main symptoms are late onset visual deficits, we discovered early impairments in hippocampus-dependent spatial memory attributable to defects in adult neurogenesis. Moreover, less connected adult-born hippocampal neurons showed a decrease in mitochondrial content. Remarkably, voluntary exercise or pharmacological treatment targeting mitochondrial dynamics restored spatial memory in DOA mice. Altogether, our study identifies a crucial role for OPA1-dependent mitochondrial functions in adult neurogenesis, and thus in hippocampal-dependent cognitive functions. More generally, our findings show that adult neurogenesis is highly sensitive to mild mitochondrial defects, generating impairments in spatial memory that can be detected at an early stage and counterbalanced by physical exercise and pharmacological targeting of mitochondrial dynamics. Thus, amplification of mitochondrial function at an early stage appears beneficial for late-onset neurodegenerative diseases.
Assuntos
Doenças Neurodegenerativas , Atrofia Óptica Autossômica Dominante , Masculino , Camundongos , Animais , Memória Espacial , Mitocôndrias/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Atrofia Óptica Autossômica Dominante/metabolismo , Hipocampo/metabolismo , Doenças Neurodegenerativas/metabolismoRESUMO
OBJECTIVE: To investigate whether long noncoding RNA H19 (lncRNA H19) induces vascular calcification by promoting calcium deposition, osteogenic differentiation and apoptosis via inhibiting the Bax inhibitor 1/optic atrophy 1 (BI-1/ OPA1) pathway. METHODS: ß-glycerophosphate and calcium chloride were used to induce calcification in rat vascular smooth muscle cells (VSMCs), and the effects of siH19, alone or in combination with BI-1 or OPA1 knockdown, on calcification of the cells were investigated. Osteogenic differentiation was assessed by measuring Runt-related transcription factor 2 (Runx-2) and bone morphogenetic protein 2 (BMP-2) expression with Western blotting, and cell apoptosis was evaluated by TUNEL staining and Western blotting. An ApoE-/- diabetic mouse model with high-fat feeding for 32 weeks were given an intraperitoneal injection of siH19, and the changes in calcium deposition in the aortic arch were examined using Alizarin red S staining and von Kossa staining. RESULTS: In rat VSMCs with calcification, the expression of lncRNA H19 was significantly increased, and the expressions of BI- 1 and OPA1 were significantly decreased. Downregulation of lncRNA H19 significantly increased the expressions of BI-1 and OPA1 proteins in the cells, and BI-1 knockdown further reduced OPA1 expression (P<0.001). The cells treated with siH19 showed total disappearance of the calcified nodules with significantly reduced expressions of Runx-2, BMP-2 and cleaved caspase-3 and a lowered cell apoptosis rate (P<0.001). Calcified nodules were again observed in the cells with lncRNA H19 knockdown combined with BI-1 or OPA1 knockdown, and the expressions of Runx-2, BMP-2, cleaved-caspase-3 and cell apoptosis rate all significantly increased (P<0.001). In the diabetic mouse model with high-fat feeding, siH19 treatment significantly reduced the calcification area and increased mRNA expressions of BI-I and OPA1 in the aortic arch. CONCLUSION: LncRNA H19 promotes vascular calcification possibly by promoting calcium deposition, osteogenic differentiation and cell apoptosis via inhibiting the BI-1/OPA1 pathway.
Assuntos
Diabetes Mellitus , Atrofia Óptica Autossômica Dominante , RNA Longo não Codificante , Calcificação Vascular , Animais , Camundongos , Ratos , Proteína X Associada a bcl-2/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Diabetes Mellitus/metabolismo , Modelos Animais de Doenças , Miócitos de Músculo Liso , Atrofia Óptica Autossômica Dominante/metabolismo , Osteogênese , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Calcificação Vascular/metabolismoRESUMO
Dominant optic atrophy is an optic neuropathy with varying clinical symptoms and progression. A severe disorder is associated with certain OPA1 mutations and includes additional symptoms for >20% of patients. This underscores the consequences of OPA1 mutations in different cellular populations, not only retinal ganglionic cells. We assessed the effects of OPA1 loss of function on oxidative metabolism and antioxidant defences using an RNA-silencing strategy in a human epithelial cell line. We observed a decrease in the mitochondrial respiratory chain complexes, associated with a reduction in aconitase activity related to an increase in reactive oxygen species (ROS) production. In response, the NRF2 (also known as NFE2L2) transcription factor was translocated into the nucleus and upregulated SOD1 and GSTP1. This study highlights the effects of OPA1 deficiency on oxidative metabolism in replicative cells, as already shown in neurons. It underlines a translational process to use cycling cells to circumvent and describe oxidative metabolism. Moreover, it paves the way to predict the evolution of dominant optic atrophy using mathematical models that consider mitochondrial ROS production and their detoxifying pathways.
Assuntos
Atrofia Óptica Autossômica Dominante , Humanos , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Mitocôndrias/metabolismo , Respiração Celular , Estresse Oxidativo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismoRESUMO
During cardiac ischemia-reperfusion, excess reactive oxygen species can damage mitochondrial, cellular and organ function. Here we show that cysteine oxidation of the mitochondrial protein Opa1 contributes to mitochondrial damage and cell death caused by oxidative stress. Oxy-proteomics of ischemic-reperfused hearts reveal oxidation of the C-terminal C786 of Opa1 and treatment of perfused mouse hearts, adult cardiomyocytes, and fibroblasts with H2O2 leads to the formation of a reduction-sensitive â¼180 KDa Opa1 complex, distinct from the â¼270 KDa one antagonizing cristae remodeling. This Opa1 oxidation process is curtailed by mutation of C786 and of the other 3 Cys residues of its C-terminal domain (Opa1TetraCys). When reintroduced in Opa1-/- cells, Opa1TetraCys is not efficiently processed into short Opa1TetraCys and hence fails to fuse mitochondria. Unexpectedly, Opa1TetraCys restores mitochondrial ultrastructure in Opa1-/- cells and protects them from H2O2-induced mitochondrial depolarization, cristae remodeling, cytochrome c release and cell death. Thus, preventing the Opa1 oxidation occurring during cardiac ischemia-reperfusion reduces mitochondrial damage and cell death induced by oxidative stress independent of mitochondrial fusion.
Assuntos
Doença da Artéria Coronariana , Traumatismo por Reperfusão Miocárdica , Atrofia Óptica Autossômica Dominante , Animais , Camundongos , Morte Celular , Cisteína/metabolismo , Peróxido de Hidrogênio , Traumatismo por Reperfusão Miocárdica/metabolismo , Atrofia Óptica Autossômica Dominante/metabolismo , Estresse OxidativoRESUMO
Inner mitochondrial membrane fusion and cristae shape depend on optic atrophy protein 1, OPA1. Mutations in OPA1 lead to autosomal dominant optic atrophy (ADOA), an important cause of inherited blindness. The Guanosin Triphosphatase (GTPase) and GTPase effector domains (GEDs) of OPA1 are essential for mitochondrial fusion; yet, their specific roles remain elusive. Intriguingly, patients carrying OPA1 GTPase mutations have a higher risk of developing more severe multisystemic symptoms in addition to optic atrophy, suggesting pathogenic contributions for the GTPase and GED domains, respectively. We studied OPA1 GTPase and GED mutations to understand their domain-specific contribution to protein function by analyzing patient-derived cells and gain-of-function paradigms. Mitochondria from OPA1 GTPase (c.870+5G>A and c.889C>T) and GED (c.2713C>T and c.2818+5G>A) mutants display distinct aberrant cristae ultrastructure. While all OPA1 mutants inhibited mitochondrial fusion, some GTPase mutants resulted in elongated mitochondria, suggesting fission inhibition. We show that the GED is dispensable for fusion and OPA1 oligomer formation but necessary for GTPase activity. Finally, splicing defect mutants displayed a posttranslational haploinsufficiency-like phenotype but retained domain-specific dysfunctions. Thus, OPA1 domain-specific mutants result in distinct impairments in mitochondrial dynamics, providing insight into OPA1 function and its contribution to ADOA pathogenesis and severity.
Assuntos
Mitocôndrias , Atrofia Óptica Autossômica Dominante , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , MutaçãoRESUMO
Dominant Optic Atrophy (DOA) is one of the most common inherited mitochondrial diseases, leading to blindness. It is caused by the chronic degeneration of the retinal ganglion cells (RGCs) and their axons forming the optic nerve. Until now, DOA has been mainly associated with genes encoding proteins involved in mitochondrial network dynamics. Using next-generation and exome sequencing, we identified for the first time heterozygous PMPCA variants having a causative role in the pathology of late-onset primary DOA in five patients. PMPCA encodes an α subunit of the mitochondrial peptidase (MPP), responsible for the cleavage and maturation of the mitochondrial precursor proteins imported from the cytoplasm into mitochondria. Recently, PMPCA has been identified as the gene responsible for Autosomal Recessive Cerebellar Ataxia type 2 (SCAR2) and another severe recessive mitochondrial disease. In this study, four PMPCA variants were identified, two are frameshifts (c.309delA and c.820delG) classified as pathogenic and two are missenses (c.1363G>A and c.1547G>A) classified with uncertain pathological significance. Functional assays on patients' fibroblasts show a hyperconnection of the mitochondrial network and revealed that frameshift variants reduced α-MPP levels, while not significantly affecting the respiratory machinery. These results suggest that alterations in mitochondrial peptidase function can affect the fusion-fission balance, a key element in maintaining the physiology of retinal ganglion cells, and consequently lead to their progressive degeneration.
Assuntos
Doenças Mitocondriais , Atrofia Óptica Autossômica Dominante , Humanos , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas Mitocondriais/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Peptídeo Hidrolases , Peptidase de Processamento MitocondrialRESUMO
OBJECTIVE: To investigate the effects of Bax inhibitor 1 (BI- 1) and optic atrophy protein 1 (OPA1) on vascular calcification (VC). METHODS: Mouse models of VC were established in ApoE-deficient (ApoE-/-) diabetic mice by high-fat diet feeding for 12 weeks followed by intraperitoneal injections with Nε-carboxymethyl-lysine for 16 weeks. ApoE-/- mice (control group), ApoE-/- diabetic mice (VC group), ApoE-/- diabetic mice with BI-1 overexpression (VC + BI-1TG group), and ApoE-/- diabetic mice with BI-1 overexpression and OPA1 knockout (VC+BI-1TG+OPA1-/- group) were obtained for examination of the degree of aortic calcification using von Kossa staining. The changes in calcium content in the aorta were analyzed using ELISA. The expressions of Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP-2) were detected using immunohistochemistry, and the expression of cleaved caspase-3 was determined using Western blotting. Cultured mouse aortic smooth muscle cells were treated with 10 mmol/L ß-glycerophosphate for 14 days to induce calcification, and the changes in BI-1 and OPA1 protein expressions were examined using Western blotting and cell apoptosis was detected using TUNEL staining. RESULTS: ApoE-/- mice with VC showed significantly decreased expressions of BI-1 and OPA1 proteins in the aorta (P=0.0044) with obviously increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P= 0.0041). Overexpression of BI-1 significantly promoted OPA1 protein expression and reduced calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 (P=0.0006). OPA1 knockdown significantly increased calcium deposition and expressions of RUNX2, BMP-2 and cleaved caspase-3 in the aorta (P=0.0007). CONCLUSION: BI-1 inhibits VC possibly by promoting the expression of OPA1, reducing calcium deposition and inhibiting osteogenic differentiation and apoptosis of the vascular smooth muscle cells.
Assuntos
Diabetes Mellitus Experimental , GTP Fosfo-Hidrolases , Proteínas de Membrana , Atrofia Óptica Autossômica Dominante , Calcificação Vascular , Animais , Apolipoproteínas E/metabolismo , Cálcio/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , GTP Fosfo-Hidrolases/biossíntese , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Osteogênese , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Dominant optic atrophy (DOA) is one of the most common type of hereditary optic atrophy. Here, we describe the generation and characterization of a human induced pluripotent stem cell (hiPSC) line of DOA patient with an OPA1 mutation. The reprogramming of this iPSC line was performed from peripheral blood mononuclear cells (PBMCs) using the non-integrative Sendai virus. The established hiPSC line retained the disease-associated mutation and showed normal karyotype, pluripotency, and differentiation capacity.
Assuntos
Células-Tronco Pluripotentes Induzidas , Atrofia Óptica Autossômica Dominante , Diferenciação Celular , Reprogramação Celular , GTP Fosfo-Hidrolases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares , Mutação/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismoRESUMO
The purpose of this study was to evaluate the neuroprotective effects of omega-3 polyunsaturated fatty acid (ω3-PUFA) supplementation in a mouse model of OPA1-associated autosomal dominant optic atrophy (ADOA). The blood level of arachidonic acid (AA) and eicosapentaenoic acid (EPA) served to adjust the treatment dosage (AA/EPA = 1.0-1.5). Eight-month-old mice were allocated to four groups (n = 20/group): the ω3-PUFA-treated Opa1enu/+, untreated Opa1enu/+, ω3-PUFA-treated wild-type and untreated wild-type groups. Treated mice received the ω3-PUFAs, EPA and docosahexaenoic acid (DHA; 5:1 ratio) by daily gavage for 4 months based on the measured AA/EPA ratio. Blood, retina and optic nerve (ON) fatty acid levels were determined by gas chromatography, and the retina and ON were histologically examined. Western blotting and/or immunohistochemistry was performed to analyse retinal mediators involved in Opa1-mutation-mediated apoptosis, inflammation and oxidative stress. Increased EPA and reduced AA levels were primarily observed predominantly in the blood and retinal tissues, and a similarly high EPA level tended to be observed in the ONs of ω3-PUFA-treated mice. Retinal ganglion cell and ON axonal densities were higher in both mouse strains upon ω3-PUFA treatment than in the corresponding untreated groups. Caspase-3 expression analysis showed fewer apoptotic retinal cells in both groups of treated mice. Decreases in inflammatory microglia and astrocytes activation and proapoptotic Bcl-2-associated X protein (Bax) expression were noted in the treated groups, with no difference in the antioxidant superoxide dismutase-2 expression. ω3-PUFA supplementation had neuroprotective effects on the retinas of Opa1enu/+ and wild-type mice via blockade of microglia and astrocytes activation and suppression of Bax and caspase-3. Our findings indicated that inhibition of oxidative stress may not be involved in ω3-PUFA-mediated neuroprotection. These novel findings support the use of ω3-PUFAs as a beneficial therapy in the occurrence of ADOA, posing the basis for future clinical trials to confirm these observations.
Assuntos
Ácidos Graxos Ômega-3 , Neuroglia , Fármacos Neuroprotetores , Atrofia Óptica Autossômica Dominante , Animais , Apoptose , Ácido Araquidônico/metabolismo , Caspase 3/metabolismo , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Ômega-3/farmacologia , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Neuroglia/metabolismo , Neuroglia/patologia , Neuroproteção , Fármacos Neuroprotetores/farmacologia , Atrofia Óptica Autossômica Dominante/tratamento farmacológico , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Retina/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
Mitochondrial dysfunction and mitophagy are often hallmarks of neurodegenerative diseases such as autosomal dominant optic atrophy (ADOA) caused by mutations in the key mitochondrial dynamics protein optic atrophy 1 (Opa1). However, the second messengers linking mitochondrial dysfunction to initiation of mitophagy remain poorly characterized. Here, we show in mammalian and nematode neurons that Opa1 mutations trigger Ca2+-dependent mitophagy. Deletion or expression of mutated Opa1 in mouse retinal ganglion cells and Caenorhabditis elegans motor neurons lead to mitochondrial dysfunction, increased cytosolic Ca2+ levels, and decreased axonal mitochondrial density. Chelation of Ca2+ restores mitochondrial density in neuronal processes, neuronal function, and viability. Mechanistically, sustained Ca2+ levels activate calcineurin and AMPK, placed in the same genetic pathway regulating axonal mitochondrial density. Our data reveal that mitophagy in ADOA depends on Ca2+-calcineurin-AMPK signaling cascade.
Assuntos
Atrofia Óptica Autossômica Dominante , Animais , Cálcio , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Mamíferos/metabolismo , Camundongos , Mitofagia/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Background: To identify and investigate the effects of a novel splicing variant, c.1444-2A>C of OPA1, on its transcript, translation, and mitochondrial function, which was found in an 8-year-old patient with dominantly inherited optic atrophy (DOA). Materials and Methods: The clinical evaluations were performed at the Eye Center. Lymphoblast cell lines were generated from the patient, mother, and a normal control with the same haplotype of mitochondrial genome. The novel variant was confirmed by Sanger sequencing. The splicing alteration of cDNA was checked by both Sanger sequencing and agarose gel. OPA1 expression was carried out by RT-PCR and Western blotting. Transmission electron microscopy was used for mitochondrial morphology. Mitochondrial functions, including the rates of oxygen consumption, ATP generation, ROS product and membrane potential were assayed in lymphoblast cells. Results: The novel OPA1 splicing variant, c.1444-2A>C, led to a deletion of the 15th exon in mRNA transcript. Approximately 50% reduction of mRNA and protein expression was present in mutant cells as compared with controls. No marked depletion of mtDNA nor mitochondrial mass was caused by the splicing variant. However, defects that the impaired capacity of OXPHOS, reduced ATP generation, increased ROS and decreased membrane potential were observed in the mutant cells, which promoted a ubiquitin-binding mitophagy instead of apoptosis. Conclusions: The novel splicing variant, c.1444-2A>C resulted in OPA1 haploinsufficiency effect on its expression and mitochondrial function without mtDNA depletion. Our findings may provide new insights into the understanding of pathophysiology of DOA.
Assuntos
DNA Mitocondrial/genética , GTP Fosfo-Hidrolases/genética , Haploinsuficiência , Mitocôndrias/patologia , Mutação , Atrofia Óptica Autossômica Dominante/patologia , Splicing de RNA , Estudos de Casos e Controles , Criança , Humanos , Masculino , Mitocôndrias/genética , Atrofia Óptica Autossômica Dominante/etiologia , Atrofia Óptica Autossômica Dominante/metabolismo , PrognósticoRESUMO
Dominant optic atrophy (DOA) is an inherited mitochondrial disease leading to specific degeneration of retinal ganglion cells (RGCs), thus compromising transmission of visual information from the retina to the brain. Usually, DOA starts during childhood and evolves to poor vision or legal blindness, affecting the central vision, whilst sparing the peripheral visual field. In 20% of cases, DOA presents as syndromic disorder, with secondary symptoms affecting neuronal and muscular functions. Twenty years ago, we demonstrated that heterozygous mutations in OPA1 are the most frequent molecular cause of DOA. Since then, variants in additional genes, whose functions in many instances converge with those of OPA1, have been identified by next generation sequencing. OPA1 encodes a dynamin-related GTPase imported into mitochondria and located to the inner membrane and intermembrane space. The many OPA1 isoforms, resulting from alternative splicing of three exons, form complex homopolymers that structure mitochondrial cristae, and contribute to fusion of the outer membrane, thus shaping the whole mitochondrial network. Moreover, OPA1 is required for oxidative phosphorylation, maintenance of mitochondrial genome, calcium homeostasis and regulation of apoptosis, thus making OPA1 the Swiss army-knife of mitochondria. Understanding DOA pathophysiology requires the understanding of RGC peculiarities with respect to OPA1 functions. Besides the tremendous energy requirements of RGCs to relay visual information from the eye to the brain, these neurons present unique features related to their differential environments in the retina, and to the anatomical transition occurring at the lamina cribrosa, which parallel major adaptations of mitochondrial physiology and shape, in the pre- and post-laminar segments of the optic nerve. Three DOA mouse models, with different Opa1 mutations, have been generated to study intrinsic mechanisms responsible for RGC degeneration, and these have further revealed secondary symptoms related to mitochondrial dysfunctions, mirroring the more severe syndromic phenotypes seen in a subgroup of patients. Metabolomics analyses of cells, mouse organs and patient plasma mutated for OPA1 revealed new unexpected pathophysiological mechanisms related to mitochondrial dysfunction, and biomarkers correlated quantitatively to the severity of the disease. Here, we review and synthesize these data, and propose different approaches for embracing possible therapies to fulfil the unmet clinical needs of this disease, and provide hope to affected DOA patients.
Assuntos
Atrofia Óptica Autossômica Dominante , Animais , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Humanos , Camundongos , Mitocôndrias , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Nervo Óptico/metabolismo , Células Ganglionares da Retina/metabolismoRESUMO
Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.
Assuntos
Proteases Dependentes de ATP/genética , ATPases Associadas a Diversas Atividades Celulares/genética , Atrofia Óptica Autossômica Dominante/genética , Atrofia Óptica Autossômica Dominante/metabolismo , Domínio AAA/genética , Adolescente , Criança , Pré-Escolar , Feminino , GTP Fosfo-Hidrolases/metabolismo , Humanos , Masculino , Metaloendopeptidases/metabolismo , Mutação de Sentido Incorreto , LinhagemRESUMO
Purpose: Retinal ganglion cells (RGCs) are susceptible to mitochondrial deficits and also the major cell type affected in patients with mutations in the OPA1 gene in autosomal dominant optic atrophy (ADOA). Here, we characterized mitochondria in RGCs in vitro from a heterozygous B6; C3-Opa1Q285STOP (Opa1+/-) mouse model to investigate mitochondrial changes underlying the pathology in ADOA. Methods: Mouse RGCs were purified from wild-type and Opa1+/- mouse retina by two-step immunopanning. The mitochondria in neurites of RGCs were labeled with MitoTracker Red for structure and motility measurement by time-lapse imaging. Mitochondrial bioenergetics were determined by the real-time measurement of oxygen consumption rate using a Seahorse XFe 96 Extracellular Flux Analyzer. Results: We observed a significant decrease in mitochondrial length in Opa1+/- RGCs with a remarkably higher proportion and density of motile mitochondria along the neurites. We also observed an increased transport velocity with a higher number of contacts between mitochondria in Opa1+/- RGC neurites. The oxygen consumption assays showed a severe impairment in basal respiration, Adenosine triphosphate-linked (ATP-linked) oxygen consumption, as well as reserve respiratory capacity, in RGCs from Opa1+/- mouse retina. Conclusions: Opa1 deficiency leads to significant fragmentation of mitochondrial morphology, activation of mitochondrial motility and impaired respiratory function in RGCs from the B6; C3-Opa1Q285STOP mouse model. This highlights the significant alterations in the intricate interplay between mitochondrial morphology, motility, and energy production in RGCs with Opa1 deficiency long before the onset of clinical symptoms of the pathology.
Assuntos
Metabolismo Energético , GTP Fosfo-Hidrolases/deficiência , Mitocôndrias/metabolismo , Mutação , Atrofia Óptica Autossômica Dominante/genética , Células Ganglionares da Retina/metabolismo , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , GTP Fosfo-Hidrolases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Atrofia Óptica Autossômica Dominante/metabolismo , Atrofia Óptica Autossômica Dominante/patologia , Células Ganglionares da Retina/patologiaRESUMO
Human iPSC line, IISHDOi006-A, was obtained from fibroblasts of a patient with Dominant Optic Atrophy (DOA) carrying a heterozygous mutation in the gene ACO2: c.1999G>A; p.Glu667Lys. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using a non-integrative methodology that involves the use of Sendai virus.