ABSTRACT
Mutations of the ADAR1 gene encoding an RNA deaminase cause severe diseases associated with chronic activation of type I interferon (IFN) responses, including Aicardi-Goutières syndrome and bilateral striatal necrosis1-3. The IFN-inducible p150 isoform of ADAR1 contains a Zα domain that recognizes RNA with an alternative left-handed double-helix structure, termed Z-RNA4,5. Hemizygous ADAR1 mutations in the Zα domain cause type I IFN-mediated pathologies in humans2,3 and mice6-8; however, it remains unclear how the interaction of ADAR1 with Z-RNA prevents IFN activation. Here we show that Z-DNA-binding protein 1 (ZBP1), the only other protein in mammals known to harbour Zα domains9, promotes type I IFN activation and fatal pathology in mice with impaired ADAR1 function. ZBP1 deficiency or mutation of its Zα domains reduced the expression of IFN-stimulated genes and largely prevented early postnatal lethality in mice with hemizygous expression of ADAR1 with mutated Zα domain (Adar1mZα/- mice). Adar1mZα/- mice showed upregulation and impaired editing of endogenous retroelement-derived complementary RNA reads, which represent a likely source of Z-RNAs activating ZBP1. Notably, ZBP1 promoted IFN activation and severe pathology in Adar1mZα/- mice in a manner independent of RIPK1, RIPK3, MLKL-mediated necroptosis and caspase-8-dependent apoptosis, suggesting a novel mechanism of action. Thus, ADAR1 prevents endogenous Z-RNA-dependent activation of pathogenic type I IFN responses by ZBP1, suggesting that ZBP1 could contribute to type I interferonopathies caused by ADAR1 mutations.
Subject(s)
Adenosine Deaminase , Interferon Type I , RNA-Binding Proteins , Adenosine Deaminase/genetics , Adenosine Deaminase/metabolism , Animals , Apoptosis , Caspase 8/metabolism , Interferon Type I/antagonists & inhibitors , Interferon Type I/immunology , Mice , Mutation , Necroptosis , RNA, Double-Stranded/metabolism , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Hereditary spastic paraplegia is a neurological condition characterized by predominant axonal degeneration in long spinal tracts, leading to weakness and spasticity in the lower limbs. The nicotinamide adenine dinucleotide (NAD+)-consuming enzyme SARM1 has emerged as a key executioner of axonal degeneration upon nerve transection and in some neuropathies. An increase in the nicotinamide mononucleotide/NAD+ ratio activates SARM1, causing catastrophic NAD+ depletion and axonal degeneration. However, the role of SARM1 in the pathogenesis of hereditary spastic paraplegia has not been investigated. Here, we report an enhanced mouse model for hereditary spastic paraplegia caused by mutations in SPG7. The eSpg7 knockout mouse carries a deletion in both Spg7 and Afg3l1, a redundant homologue expressed in mice but not in humans. The eSpg7 knockout mice recapitulate the phenotypic features of human patients, showing progressive symptoms of spastic-ataxia and degeneration of axons in the spinal cord as well as the cerebellum. We show that the lack of SPG7 rewires the mitochondrial proteome in both tissues, leading to an early onset decrease in mito-ribosomal subunits and a remodelling of mitochondrial solute carriers and transporters. To interrogate mechanisms leading to axonal degeneration in this mouse model, we explored the involvement of SARM1. Deletion of SARM1 delays the appearance of ataxic signs, rescues mitochondrial swelling and axonal degeneration of cerebellar granule cells and dampens neuroinflammation in the cerebellum. The loss of SARM1 also prevents endoplasmic reticulum abnormalities in long spinal cord axons, but does not halt the degeneration of these axons. Our data thus reveal a neuron-specific interplay between SARM1 and mitochondrial dysfunction caused by lack of SPG7 in hereditary spastic paraplegia.
Subject(s)
Spastic Paraplegia, Hereditary , Animals , Humans , Mice , Armadillo Domain Proteins/genetics , ATPases Associated with Diverse Cellular Activities , Axons/pathology , Cerebellum , Cytoskeletal Proteins/genetics , Metalloendopeptidases/genetics , Metalloendopeptidases/metabolism , NAD , Spastic Paraplegia, Hereditary/geneticsABSTRACT
Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca2+ homeostasis.
Subject(s)
Calcium Channels/metabolism , Cerebellum/metabolism , Corpus Striatum/metabolism , Hippocampus/metabolism , Metalloendopeptidases/genetics , Mitochondria/metabolism , Neurons/metabolism , ATP-Dependent Proteases/genetics , ATP-Dependent Proteases/metabolism , ATPases Associated with Diverse Cellular Activities , Animals , Calcium/metabolism , Calcium Channels/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Cell Death , Cerebellum/pathology , Corpus Striatum/pathology , Gene Expression Regulation , HEK293 Cells , Hippocampus/pathology , Homeostasis/genetics , Humans , Ion Transport , Metalloendopeptidases/deficiency , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mitochondria/pathology , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Permeability Transition Pore , Neurons/pathology , Protein Interaction Mapping , Signal TransductionABSTRACT
Mitochondrial oxidative phosphorylation (OXPHOS) fuels cellular ATP demands. OXPHOS defects lead to severe human disorders with unexplained tissue specific pathologies. Mitochondrial gene expression is essential for OXPHOS biogenesis since core subunits of the complexes are mitochondrial-encoded. COX14 is required for translation of COX1, the central mitochondrial-encoded subunit of complex IV. Here we describe a COX14 mutant mouse corresponding to a patient with complex IV deficiency. COX14M19I mice display broad tissue-specific pathologies. A hallmark phenotype is severe liver inflammation linked to release of mitochondrial RNA into the cytosol sensed by RIG-1 pathway. We find that mitochondrial RNA release is triggered by increased reactive oxygen species production in the deficiency of complex IV. Additionally, we describe a COA3Y72C mouse, affected in an assembly factor that cooperates with COX14 in early COX1 biogenesis, which displays a similar yet milder inflammatory phenotype. Our study provides insight into a link between defective mitochondrial gene expression and tissue-specific inflammation.
Subject(s)
Electron Transport Complex IV , Inflammation , Liver , Oxidative Phosphorylation , Reactive Oxygen Species , Animals , Reactive Oxygen Species/metabolism , Mice , Liver/metabolism , Liver/pathology , Inflammation/metabolism , Inflammation/genetics , Inflammation/pathology , Electron Transport Complex IV/metabolism , Electron Transport Complex IV/genetics , Mitochondria/metabolism , Humans , RNA, Mitochondrial/genetics , RNA, Mitochondrial/metabolism , Male , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Protein Biosynthesis , Mice, Inbred C57BL , DEAD-box RNA Helicases/metabolism , DEAD-box RNA Helicases/genetics , Female , Mutation , DEAD Box Protein 58 , Membrane Proteins , Cyclooxygenase 1ABSTRACT
OBJECTIVE: The ever-increasing number of genetically engineered mouse models highlights the need for efficient archiving and distribution of these lines. Sperm cryopreservation has become the preferred technique for the majority of these models due to its low requirement of costs, time, and experimental animals. Yet, current in vitro fertilization (IVF) protocols either exhibit decreased fertilization efficiency for the most popular C57BL/6 strain, as recently demonstrated by us, or require costly and difficult-to-prepare media, respectively. As a result, we previously developed SEcuRe, a modified IVF protocol with low costs and high fertilization efficiency. The popular basal fertilization medium, Cook's® proprietary "Research vitro fert" (RVF), used in this protocol has recently been discontinued. As a result, the application of the SEcuRe approach and other IVF protocols employing this medium has been severely limited. RESULTS: Here we show that human tubal fluid (HTF), a popular and widely available medium with a known composition, can be used as a basal fertilization medium instead of RVF. Comparison of RVF and HTF during 58 independent SEcuRe IVFs with cryopreserved C57BL/6 sperm revealed equal fertilization and live birth rates. In addition, we demonstrate that HTF has a substantially extended shelf-life by utilizing commercial HTF that was six months past its expiration date, yet did not affect fertilization during IVF or subsequent embryo development. This finding not only increases the economic value of our modified method, but also validates it once more. Our results demonstrate that common, shelf-life extended HTF can be used in SEcuRe IVF in place of now-discontinued RVF medium and ensure the applicability of the method, which we since termed SEcuRe 2.0. Our modified SEcuRe 2.0 strategy will assist researchers to efficiently archive and distribute genetically engineered mouse models in a cost-effective, easily adaptable, and 3R-compliant manner with minimal animal use.
Subject(s)
Fertilization in Vitro , Semen , Male , Humans , Animals , Mice , Mice, Inbred C57BL , Reproductive Techniques, Assisted , FertilizationABSTRACT
CRISPR-mediated genome editing has undoubtedly revolutionized genetic engineering of animals. With the ability for virtually unlimited modification of almost any genome it is easy to forget which amazing discoveries paved the way for this ground-breaking technology. Here, we summarize the history of genome editing platforms, starting from enhanced integration of foreign DNA by meganuclease-mediated double-strand breaks to CRISPR/Cas9, the leading technology to date, and its re-engineered variants.
Subject(s)
CRISPR-Cas Systems , Gene Editing , Animals , Genetic EngineeringABSTRACT
De novo mutations in voltage- and ligand-gated channels have been associated with an increasing number of cases of developmental and epileptic encephalopathies, which often fail to respond to classic antiseizure medications. Here, we examine two knock-in mouse models replicating de novo sequence variations in the human HCN1 voltage-gated channel gene, p.G391D and p.M153I (Hcn1G380D/+ and Hcn1M142I/+ in mouse), associated with severe drug-resistant neonatal- and childhood-onset epilepsy, respectively. Heterozygous mice from both lines displayed spontaneous generalized tonic-clonic seizures. Animals replicating the p.G391D variant had an overall more severe phenotype, with pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons. In line with clinical reports from patients with pathogenic HCN1 sequence variations, administration of the antiepileptic Na+ channel antagonists lamotrigine and phenytoin resulted in the paradoxical induction of seizures in both mouse lines, consistent with an impairment in inhibitory neuron function. We also show that these variants can render HCN1 channels unresponsive to classic antagonists, indicating the need to screen mutated channels to identify novel compounds with diverse mechanism of action. Our results underscore the necessity of tailoring effective therapies for specific channel gene variants, and how strongly validated animal models may provide an invaluable tool toward reaching this objective.
Subject(s)
Brain Diseases , Ligand-Gated Ion Channels , Animals , Anticonvulsants , Brain Diseases/genetics , Child , Humans , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/genetics , Lamotrigine , Mice , Phenytoin , Potassium Channels/genetics , Seizures/drug therapy , Seizures/geneticsABSTRACT
The advent of genome editing tools like CRISPR/Cas has substantially increased the number of genetically engineered mouse models in recent years. In support of refinement and reduction, sperm cryopreservation is advantageous compared to embryo freezing for archiving and distribution of such mouse models. The in vitro fertilization using cryopreserved sperm from the most widely used C57BL/6 strain has become highly efficient in recent years due to several improvements of the procedure. However, purchase of the necessary media for routine application of the current protocol poses a constant burden on budgetary constraints. In-house media preparation, instead, is complex and requires quality control of each batch. Here, we describe a cost-effective and easily adaptable approach for in vitro fertilization using cryopreserved C57BL/6 sperm. This is mainly achieved by modification of an affordable commercial fertilization medium and a step-by-step description of all other necessary reagents. Large-scale comparison of fertilization rates from independent lines of genetically engineered C57BL/6 mice upon cryopreservation and in vitro fertilization with our approach demonstrated equal or significantly superior fertilization rates to current protocols. Our novel SEcuRe (Simple Economical set-up for Rederivation) method provides an affordable, easily adaptable and harmonized protocol for highly efficient rederivation using cryopreserved C57BL/6 sperm for a broad application of colony management in the sense of the 3Rs.
Subject(s)
Cryopreservation/methods , Semen Preservation/methods , Animals , Costs and Cost Analysis , Cryopreservation/economics , Cryopreservation/veterinary , Female , Fertilization in Vitro/methods , Fertilization in Vitro/veterinary , Male , Mice , Mice, Inbred C57BL , Semen Preservation/economics , Semen Preservation/veterinaryABSTRACT
Obesity and type 2 diabetes mellitus are global emergencies and long noncoding RNAs (lncRNAs) are regulatory transcripts with elusive functions in metabolism. Here we show that a high fraction of lncRNAs, but not protein-coding mRNAs, are repressed during diet-induced obesity (DIO) and refeeding, whilst nutrient deprivation induced lncRNAs in mouse liver. Similarly, lncRNAs are lost in diabetic humans. LncRNA promoter analyses, global cistrome and gain-of-function analyses confirm that increased MAFG signaling during DIO curbs lncRNA expression. Silencing Mafg in mouse hepatocytes and obese mice elicits a fasting-like gene expression profile, improves glucose metabolism, de-represses lncRNAs and impairs mammalian target of rapamycin (mTOR) activation. We find that obesity-repressed LincIRS2 is controlled by MAFG and observe that genetic and RNAi-mediated LincIRS2 loss causes elevated blood glucose, insulin resistance and aberrant glucose output in lean mice. Taken together, we identify a MAFG-lncRNA axis controlling hepatic glucose metabolism in health and metabolic disease.
Subject(s)
Diabetes Mellitus, Type 2/genetics , Glucose/metabolism , Liver/metabolism , MafG Transcription Factor/genetics , Obesity/genetics , RNA, Long Noncoding/genetics , Repressor Proteins/genetics , Aged , Animals , Diabetes Mellitus, Type 2/metabolism , Humans , MafG Transcription Factor/metabolism , Male , Mice , Middle Aged , Obesity/metabolism , RNA, Long Noncoding/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Repressor Proteins/metabolism , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/metabolismABSTRACT
Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.
Subject(s)
CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Electroporation/methods , Gene Editing/methods , Zygote/metabolism , Animals , Electroporation/economics , Electroporation/instrumentation , Endonucleases/genetics , Endonucleases/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , RNA, Guide, Kinetoplastida/genetics , RNA, Guide, Kinetoplastida/metabolism , Zygote/growth & developmentABSTRACT
Proteolytic cleavage of the dynamin-like guanosine triphosphatase OPA1 in mitochondria is emerging as a central regulatory hub that determines mitochondrial morphology under stress and in disease. Stress-induced OPA1 processing by OMA1 triggersmitochondrial fragmentation, which is associated with mitophagy and apoptosis in vitro. Here, we identify OMA1 as a critical regulator of neuronal survival in vivo and demonstrate that stress-induced OPA1 processing by OMA1 promotes neuronal death and neuroinflammatory responses. Using mice lacking prohibitin membrane scaffolds as a model of neurodegeneration, we demonstrate that additional ablation of Oma1 delays neuronal loss and prolongs lifespan. This is accompanied by the accumulation of fusion-active, long OPA1 forms, which stabilize the mitochondrial genome but do not preserve mitochondrial cristae or respiratory chain supercomplex assembly in prohibitin-depleted neurons. Thus, long OPA1 forms can promote neuronal survival independently of cristae shape, whereas stress-induced OMA1 activation and OPA1 cleavage limit mitochondrial fusion and promote neuronal death.