Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum in a degenerating neuron.rd1 mouse ͉ photoreceptor ͉ retinal stem cells R etinitis pigmentosa (RP) is a form of retinal degeneration resulting from rod photoreceptor cell death and leading to blindness. Despite the remarkable genetic heterogeneity of this disease, photoreceptors undergo a common mode of cell death: apoptosis. An autosomal recessive form of RP is caused by mutations in the rod-specific -catalytic subunit of the phosphodiesterase gene PDE6B (1). The naturally occurring retinal degeneration (rd1) mouse is the animal model for this type of RP (2). The rd1 mouse has elevated levels of cGMP (3, 4), and this elevation results in elevated intracellular calcium (5). Ca 2ϩ concentration within the cytosol as well as Ca 2ϩ tides and ebbs within various organelles, such as mitochondria, nucleus, and endoplasmic reticulum (ER), are important in regulating many cellular functions such as neuronal survival or cell death.There are instances, most notably after Ca 2ϩ overload, in which the cell-death pathway elicited differs from classical caspasemediated apoptosis. Calpains are cysteine proteases activated by calcium during apoptotic processes (6). Calpain I and II (-and m-calpain) are expressed in the retina (7), and recent reports showed activation of calpain and cathepsin D in rd1 mice (5, 8). Several proteins are known targets of calpain protease activity, such as caspase-12 and apoptosis-inducing factor (AIF). Caspase-12, localized to the ER (9), can be activated by m-calpain in the presence of the pancaspase inhibitor zVAD.fmk (10). Interestingly, caspase-12 has been linked to neuronal degeneration in neurotoxicity caused by amyloid- protein (9), by prion protein (11), and in animal models of ALS (12). The cleaved active form of caspase-12 participates to the apoptotic event by translocation to the nucleus (13); however, it is unclear whether caspase-12 can induce chromatin fragmentation. AIF also directly translocates to the nucleus to execute DNA fragmentation that culminates in cell death (14). The translocation of AIF from mitochondria to the nucleus has been implicated in neuronal d...
Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.
Mitochondrial diseases ( MD s) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the micro RNA s miR‐181a and miR‐181b (miR‐181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these mi RNA s enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR‐181a/b inactivation in different animal models of MD s, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR‐181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR‐181a/b regulate mitochondrial homeostasis and that these mi RNA s may be effective gene‐independent therapeutic targets for MD s characterized by neuronal degeneration.
Oral-facial-digital type I (OFDI) syndrome is an X-linked male lethal developmental disorder. It is ascribed to ciliary dysfunction and characterized by malformation of the face, oral cavity, and digits. Conditional inactivation using different Cre lines allowed us to study the role of the Ofd1 transcript in limb development. Immunofluorescence and ultrastructural studies showed that Ofd1 is necessary for correct ciliogenesis in the limb bud but not for cilia outgrowth, in contrast to what was previously shown for the embryonic node. Mutants with mesenchymal Ofd1 inactivation display severe polydactyly with loss of antero-posterior (A/P) digit patterning and shortened long bones. Loss of digit identity was found to be associated with a progressive loss of Shh signaling and an impaired processing of Gli3, whereas defects in limb outgrowth were due to defective Ihh signaling and to mineralization defects during endochondral bone formation. Our data demonstrate that Ofd1 plays a role in regulating digit number and identity during limb and skeletal patterning increasing insight on the functional role of primary cilia during development.
Non-coding RNAs provide additional regulatory layers to gene expression as well as the potential to being exploited as therapeutic tools. Non-coding RNA-based therapeutic approaches have been attempted in dominant diseases, however their use for treatment of genetic diseases caused by insufficient gene dosage is currently more challenging. SINEUPs are long antisense non-coding RNAs that up-regulate translation in mammalian cells in a gene-specific manner, although, so far evidence of SINEUP efficacy has only been demonstrated in in vitro systems. We now show that synthetic SINEUPs effectively and specifically increase protein levels of a gene of interest in vivo. We demonstrated that SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human disorder leading to amelioration of the disease phenotype. Our results demonstrate that SINEUPs act through mechanisms conserved among vertebrates and that SINEUP technology can be successfully applied in vivo as a new research and therapeutic tool for gene-specific up-regulation of endogenous functional proteins.
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