optical-coherence-tomography-imaging-mouse-retinal-ganglion-cells

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells <em>In Vivo</em>

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

INSERM U1051, Institut of Neurosciences of Montpellier

The mitochondrial respiratory chain (RC) results from the expression of both mitochondrial and nuclear genes. The number of disease-causing mutations in nuclear genes is steadily growing and mitochondrial DNA (mtDNA) deletions and mutations account for no more than 15-20% of pediatric patients. Unfortunately, the disease-causing mutations have been identified for only a small number of patients. Thus, elucidating the genetic bases of RC is both essential for genetic diagnosis of patients and for fundamental knowledge of these disorders. The molecular diagnostics of mitochondrial disorders come under both genetic diagnosis and research. Indeed, identification of a new gene in a specific patient allows to perform genetic diagnosis in other families and identification of mutations in already known disease-causing genes allows to constitute a cohort of patients for further functional studies. Thus, elucidating the genetic bases of RC deficiency is an essential task that needs the use of several appropriate strategies. Fine phenotypage of patients and candidate gene screening is the first step for the constitution of a well-characterized cohort of patients. Genetic mapping has to be used in large families. This approach is greatly enhanced in the case of consanguineous families. The consanguinity of the parents should also lead to test genetic markers surrounding the gene loci rather than to directly sequence several candidate genes. However, the main problem is encountered in the cases of sporadic cases for which no genetic approaches can be developed. In these cases, functional complementation by human chromosomes or cDNA is the only presently available strategy.

Molecular diagnostics of mitochondrial disorders.

The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.

MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.

Defects in NADH:ubiquinone oxidoreductase (complex I), the largest complex of the mitochondrial respiratory chain, account for most cases of respiratory chain deficiency in human. Complex I contains at least 45 subunits, 7 of which are encoded by mitochondrial DNA (mtDNA). Here we report a novel 10197G>A mutation of the ND3 gene in three unrelated families with Leigh syndrome (LS) or dystonia. Variable degrees of heteroplasmy were found in all tissues tested and a high percentage of mutant mtDNA was observed in muscle. The 10197G>A mutation modifies a hydrophobic alanine residue into a hydrophilic threonine (A47T) in a highly conserved domain of ND3 subunit. Furthermore, this defect could be transferred along with the mutant mtDNAs to rho degrees lymphoblastoid cells in cybrid experiments. However, nuclear modifier genes may also play a role in the phenotypic expression and severity of the 10197G>A mutation. The association of the 10197G>A ND3 mutation with an isolated biochemical defect involving complex I and the discovery of the 10197G>A mutation with a similar phenotype in three unrelated families establish its pathogenicity and demonstrate that the amino acid position A47 is important for the function of complex I. These results show that the 10197G>A mutation in the mitochondrial ND3 gene should be considered as a common mtDNA mutation responsible for LS and dystonia.

A novel recurrent mitochondrial DNA mutation in ND3 gene is associated with isolated complex I deficiency causing Leigh syndrome and dystonia.

To determine the actual incidence of mitochondrial DNA (mtDNA) depletion syndrome in multiple respiratory chain deficiency.

Mitochondrial DNA depletion is a prevalent cause of multiple respiratory chain deficiency in childhood.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS; MIM 251880) is a prevalent cause of oxidative phosphorylation disorders characterized by a reduction in mtDNA copy number. The hitherto recognized disease mechanisms alter either mtDNA replication (POLG (ref. 1)) or the salvage pathway of mitochondrial deoxyribonucleosides 5'-triphosphates (dNTPs) for mtDNA synthesis (DGUOK (ref. 2), TK2 (ref. 3) and SUCLA2 (ref. 4)). A last gene, MPV17 (ref. 5), has no known function. Yet the majority of cases remain unexplained. Studying seven cases of profound mtDNA depletion (1-2% residual mtDNA in muscle) in four unrelated families, we have found nonsense, missense and splice-site mutations and in-frame deletions of the RRM2B gene, encoding the cytosolic p53-inducible ribonucleotide reductase small subunit. Accordingly, severe mtDNA depletion was found in various tissues of the Rrm2b-/- mouse. The mtDNA depletion triggered by p53R2 alterations in both human and mouse implies that p53R2 has a crucial role in dNTP supply for mtDNA synthesis.

Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion.

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by a reduction in mtDNA copy number. Several nuclear genes have been shown to account for these severe oxidative phosphorylation disorders, but the disease-causing mutations remain largely unknown.

Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion.

Some cases of mitochondrial diseases are due to mitochondrial DNA instability: multiple deletions or depletions. These anomalies are responsible for a mitochondrial respiratory chain impairment leading to various clinical involvements ranging from mild features with multiple mtDNA deletions to severe organ failure and premature death caused by mtDNA depletions. Both, deletions and depletions share an important and common feature between these two specificities: indeed, both are expressed in a tissue-specific manner.

[Mitochondrial genome instability and associated diseases].

Eukaryotic cells harbor a small multiploid mitochondrial genome, organized in nucleoids spread within the mitochondrial network. Maintenance and distribution of mitochondrial DNA (mtDNA) are essential for energy metabolism, mitochondrial lineage in primordial germ cells, and to prevent mtDNA instability, which leads to many debilitating human diseases. Mounting evidence suggests that the actors of the mitochondrial network dynamics, among which is the intramitochondrial dynamin OPA1, might be involved in these processes. Here, using siRNAs specific to OPA1 alternate spliced exons, we evidenced that silencing of the OPA1 variants including exon 4b leads to mtDNA depletion, secondary to inhibition of mtDNA replication, and to marked alteration of mtDNA distribution in nucleoid and nucleoid distribution throughout the mitochondrial network. We demonstrate that a small hydrophobic 10-kDa peptide generated by cleavage of the OPA1-exon4b isoform is responsible for this process and show that this peptide is embedded in the inner membrane and colocalizes and coimmunoprecipitates with nucleoid components. We propose a novel synthetic model in which a peptide, including two trans-membrane domains derived from the N terminus of the OPA1-exon4b isoform in vertebrates or from its ortholog in lower eukaryotes, might contribute to nucleoid attachment to the inner mitochondrial membrane and promotes mtDNA replication and distribution. Thus, this study places OPA1 as a direct actor in the maintenance of mitochondrial genome integrity.

OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution.

Dominant optic atrophy is a rare inherited optic nerve degeneration caused by mutations in the mitochondrial fusion gene OPA1. Recently, the clinical spectrum of dominant optic atrophy has been extended to frequent syndromic forms, exhibiting various degrees of neurological and muscle impairments frequently found in mitochondrial diseases. Although characterized by a specific loss of retinal ganglion cells, the pathophysiology of dominant optic atrophy is still poorly understood. We generated an Opa1 mouse model carrying the recurrent Opa1(delTTAG) mutation, which is found in 30% of all patients with dominant optic atrophy. We show that this mouse displays a multi-systemic poly-degenerative phenotype, with a presentation associating signs of visual failure, deafness, encephalomyopathy, peripheral neuropathy, ataxia and cardiomyopathy. Moreover, we found premature age-related axonal and myelin degenerations, increased autophagy and mitophagy and mitochondrial supercomplex instability preceding degeneration and cell death. Thus, these results support the concept that Opa1 protects against neuronal degeneration and opens new perspectives for the exploration and the treatment of mitochondrial diseases.

The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse.

The maintenance of mitochondrial genome integrity is a major challenge for cells to sustain energy production by respiration.

Why mitochondria must fuse to maintain their genome integrity.

OPA1 mutations are responsible for more than half of autosomal dominant optic atrophy (ADOA), a blinding disease affecting the retinal ganglion neurons. In most patients the clinical presentation is restricted to the optic nerve degeneration, albeit in 20% of them, additional neuro-sensorial symptoms might be associated to the loss of vision, as frequently encountered in mitochondrial diseases. This study describes clinical and neuroradiological features of OPA1 patients.

Neuroradiological findings expand the phenotype of OPA1-related mitochondrial dysfunction.

The myelin sheath that covers a large amount of neurons is critical for their homeostasis, and myelinating glia mitochondria have recently been shown to be essential for neuron survival. However morphological and physiological properties of these organelles remain elusive. Here we report a method to analyze mitochondrial dynamics and morphology in myelinating Schwann cells of living mice using viral transduction and time-lapse multiphoton microscopy. We describe the distribution, shape, size and dynamics of mitochondria in live cells. We also report mitochondrial alterations in Opa1(delTTAG) mutant mice cells at presymptomatic stages, suggesting that mitochondrial defects in myelin contribute to OPA1 related neuropathy and represent a biomarker for the disease.

In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath.

Mutations in the Optic Atrophy 1 gene (OPA1) were first identified in 2000 as the main cause of Dominant Optic Atrophy, a disease specifically affecting the retinal ganglion cells and the optic nerve. Since then, an increasing number of symptoms involving the central, peripheral and autonomous nervous systems, with considerable variations of age of onset and severity, have been reported in OPA1 patients. This variety of phenotypes is attributed to differences in the effects of OPA1 mutations, to the mode of inheritance, which may be mono- or bi-allelic, and eventually to somatic mitochondrial DNA mutations. The diversity of the pathophysiological mechanisms involved in OPA1-related disorders is linked to the crucial role played by OPA1 in the maintenance of mitochondrial structure, genome and function. The neurological expression of these disorders highlights the importance of mitochondrial dynamics in neuronal processes such as dendritogenesis, axonal transport, and neuronal survival. Thus, OPA1-related disorders may serve as a paradigm in the wider context of neurodegenerative syndromes, particularly for the development of novel therapeutic strategies against these diseases.

OPA1-related disorders: Diversity of clinical expression, modes of inheritance and pathophysiology.

OPA1 mutations are responsible for autosomal dominant optic atrophy (ADOA), a progressive blinding disease characterized by retinal ganglion cell (RGC) degeneration and large phenotypic variations, the underlying mechanisms of which are poorly understood. OPA1 encodes a mitochondrial protein with essential biological functions, its main roles residing in the control of mitochondrial membrane dynamics as a pro-fusion protein and prevention of apoptosis. Considering recent findings showing the importance of the mitochondrial fusion process and the involvement of OPA1 in controlling steroidogenesis, we tested the hypothesis of deregulated steroid production in retina due to a disease-causing OPA1 mutation and its contribution to the visual phenotypic variations. Using the mouse model carrying the human recurrent OPA1 mutation, we disclosed that Opa1 haploinsufficiency leads to very high circulating levels of steroid precursor pregnenolone in females, causing an early-onset vision loss, abolished by ovariectomy. In addition, steroid production in retina is also increased which, in conjunction with high circulating levels, impairs estrogen receptor expression and mitochondrial respiratory complex IV activity, promoting RGC apoptosis in females. We further demonstrate the involvement of Muller glial cells as increased pregnenolone production in female cells is noxious and compromises their role in supporting RGC survival. In parallel, we analyzed ophthalmological data of a multicentre OPA1 patient cohort and found that women undergo more severe visual loss at adolescence and greater progressive thinning of the retinal nerve fibres than males. Thus, we disclosed a gender-dependent effect on ADOA severity, involving for the first time steroids and Müller glial cells, responsible for RGC degeneration.

Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy.

Recent data suggests the involvement of mitochondrial dynamics in cardiac ischemia/reperfusion (I/R) injuries. Whilst excessive mitochondrial fission has been described as detrimental, the role of fusion proteins in this context remains uncertain.

Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model.

Dominant optic atrophy (MIM No. 165500) is a blinding condition related to mutations in OPA1, a gene encoding a large GTPase involved in mitochondrial inner membrane dynamics. Although several mouse models mimicking the disease have been developed, the pathophysiological mechanisms responsible for retinal ganglion cell degeneration remain poorly understood.

Article	Year
Molecular diagnostics of mitochondrial disorders. Biochimica et biophysica acta\| PubMed ID: 15576044	2004
MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion. Nature genetics\| PubMed ID: 16582910	2006
A novel recurrent mitochondrial DNA mutation in ND3 gene is associated with isolated complex I deficiency causing Leigh syndrome and dystonia. American journal of medical genetics. Part A\| PubMed ID: 17152068	2007
Mitochondrial DNA depletion is a prevalent cause of multiple respiratory chain deficiency in childhood. The Journal of pediatrics\| PubMed ID: 17452231	2007
Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion. Nature genetics\| PubMed ID: 17486094	2007
Twinkle helicase (PEO1) gene mutation causes mitochondrial DNA depletion. Annals of neurology\| PubMed ID: 17722119	2007
[Mitochondrial genome instability and associated diseases]. Medecine sciences : M/S\| PubMed ID: 20188049	2010
OPA1 links human mitochondrial genome maintenance to mtDNA replication and distribution. Genome research\| PubMed ID: 20974897	2011
The human OPA1delTTAG mutation induces premature age-related systemic neurodegeneration in mouse. Brain : a journal of neurology\| PubMed ID: 23250881	2012
Why mitochondria must fuse to maintain their genome integrity. Antioxidants & redox signaling\| PubMed ID: 23350575	2013
Neuroradiological findings expand the phenotype of OPA1-related mitochondrial dysfunction. Journal of the neurological sciences\| PubMed ID: 25641387	2015
In vivo time-lapse imaging of mitochondria in healthy and diseased peripheral myelin sheath. Mitochondrion\| PubMed ID: 26031781	2015
OPA1-related disorders: Diversity of clinical expression, modes of inheritance and pathophysiology. Neurobiology of disease\| PubMed ID: 26311407	2016
Increased steroidogenesis promotes early-onset and severe vision loss in females with OPA1 dominant optic atrophy. Human molecular genetics\| PubMed ID: 27260406	2016
Increase in Cardiac Ischemia-Reperfusion Injuries in Opa1+/- Mouse Model. PloS one\| PubMed ID: 27723783	2016
Targeted Metabolomics Reveals Early Dominant Optic Atrophy Signature in Optic Nerves of Opa1delTTAG/+ Mice. Investigative ophthalmology & visual science\| PubMed ID: 28159969	2017

Emmanuelle Sarzi

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