Recent advances in, and challenges of, designing OMA1 drug screens.

The proteases of the mitochondrial inner membrane are challenging yet highly desirable drug targets for complex, multifactorial diseases prevalent mainly in the elderly. Among them, OMA1 with its substrates OPA1 and DELE1 safeguards mitochondrial homeostasis at the intersection of energy metabolism and apoptosis, which may have relevance for neurodegeneration, malignancy and heart failure, among other diseases. Little is known about OMA1. Its structure has not been solved and we are just beginning to understand the enzyme's context-dependent regulation. OMA1 appears dormant under physiological conditions as judged by OPA1's processing pattern. The protease is rapidly activated, however, when cells experience stress or undergo apoptosis. Intriguingly, genetic OMA1 ablation can delay or even prevent apoptosis in animal models for diseases that can be broadly categorized as ischemia-reperfusion related disorders. Three groups have reported their efforts implementing OMA1 drug screens. This article reviews some of the technical challenges encountered in these assays and highlights what can be learned for future screening campaigns, and about the OMA1 protease more broadly. OMA1 does not exists in a vacuum and potent OMA1 inhibitors are needed to tease apart OMA1's intricate interactions with the other mitochondrial proteases and enzymes. Furthermore, OMA1 inhibitors hold the promise of becoming a new class of cytoprotective medicines for disorders influenced by dysfunctional mitochondria, such as heart failure or Alzheimer's Disease.

OMA1 High-Throughput Screen Reveals Protease Activation by Kinase Inhibitors.

Mitochondrial proteases are interesting but challenging drug targets for multifactorial diseases, such as neurodegeneration and cancer. The mitochondrial inner membrane protease OMA1 is a bona fide drug target for heart failure supported by data from human linkage analysis and animal disease models, but presumably relevant for more indications. OMA1 acts at the intersection of energy metabolism and stress signaling. The protease cleaves the structural protein OPA1, which organizes the cristae, as well as the signaling peptide DELE1, which can stimulate the integrated stress response. OMA1 shows little activity under physiological conditions but hydrolyzes OPA1 in mitochondria destined for mitophagy and during apoptosis. Little is known about OMA1, its structure has not been solved, let alone its context-dependent regulation. Autocatalytic processing and the lack of OMA1 inhibitors are thereby creating the biggest roadblocks. This study introduces a scalable, cellular OMA1 protease assay suitable for high-throughput drug screening. The assay utilizes an engineered luciferase targeted to the inner membrane as artificial OMA1 substrate, whereby the reporter signal inversely correlates to OMA1 activity. Testing different screening protocols and sampling different compound collections validated the reporter and demonstrated that both OMA1 activators as well as OMA1 inhibitors can be identified with the assay. Ten kinase-targeted cancer drugs triggered OMA1 in the assays, which suggests─considering cardiotoxicity as a rather common side-effect of this class of drugs─cross-reactivity with the OMA1 pathway.

Tau phosphorylation and OPA1 proteolysis are unrelated events: Implications for Alzheimer's Disease.

The neuropathological hallmarks of Alzheimer's Disease are plaques and neurofibrillary tangles. Yet, Alzheimer's is a complex disease with many contributing factors, such as energy-metabolic changes, which have been documented in autopsy brains from individuals with Alzheimer's and animal disease models alike. One conceivable explanation is that the interplay of age-related extracellular and intracellular alterations pertaining to Alzheimer's, such as cerebrovascular changes, protein aggregates and inflammation, evoke a mitochondrial response. However, it is not clear if and how mitochondria can contribute to Alzheimer's pathophysiology. This study focuses on one particular aspect of this question by investigating the functional interaction between the microtubule-associated protein tau and the mitochondrial inner membrane fusion machinery, which shows alterations in Alzheimer's brains. OPA1 is an essential inner membrane-fusion protein regulated by the two membrane proteases OMA1 and YME1L1. Assessment of OPA1 proteolysis-usually found in dividing mitochondria-and posttranslational tau modifications in mouse and human neuroblastoma cells under different experimental conditions clarified the relationship between these two pathways: OPA1 hydrolysis and phosphorylation or dephosphorylation of tau may coincide, but are not causally related. OPA1 cleavage did not alter tau's phosphorylation pattern. Conversely, tau's phosphorylation state did not induce nor correlate with OPA1 proteolysis. These results irrefutably demonstrate that there is no direct functional interaction between posttranslational tau modifications and the regulation of the OMA1-OPA1 pathway, which implies a common root cause modulating both pathways in Alzheimer's.

OMA1-An integral membrane protease?

OMA1 is a mitochondrial protease. Among its substrates are DELE1, a signaling peptide, which can elicit the integrated stress response, as well as the membrane-shaping dynamin-related GTPase OPA1, which can drive mitochondrial outer membrane permeabilization. OMA1 is dormant under physiological conditions but rapidly activated upon mitochondrial stress, such as loss of membrane potential or excessive reactive oxygen species. Accordingly, OMA1 was found to be activated in a number of disease conditions, including cancer and neurodegeneration. OMA1 has a predicted transmembrane domain and is believed to be tethered to the mitochondrial inner membrane. Yet, its structure has not been resolved and its context-dependent regulation remains obscure. Here, I review the literature with focus on OMA1's biochemistry. I provide a good homology model of OMA1's active site with a root-mean-square deviation of 0.9 Šand a DALI Z-score of 19.8. And I build a case for OMA1 actually being an integral membrane protease based on OMA1's role in the generation of small signaling peptides, its functional overlap with PARL, and OMA1's homology with ZMPSTE24. The refined understanding of this important enzyme can help with the design of tool compounds and development of chemical probes in the future.

Targeted OMA1 therapies for cancer.

The mitochondrial inner membrane proteins OMA1 and OPA1 belong to the BAX/BAK1-dependent apoptotic signaling pathway, which can be regulated by tumor protein p53 and the prohibitins PHB and PHB2 in the context of neoplastic disease. For the most part these proteins have been studied separate from each other. Here, I argue that the OMA1 mechanism of action represents the missing link between p53 and cytochrome c release. The mitochondrial fusion protein OPA1 is cleaved by OMA1 in a stress-dependent manner generating S-OPA1. Excessive S-OPA1 can facilitate outer membrane permeabilization upon BAX/BAK1 activation through its membrane shaping properties. p53 helps outer membrane permeabilization in a 2-step process. First, cytosolic p53 activates BAX/BAK1 at the mitochondrial surface. Then, in a second step, p53 binds to prohibitin thereby releasing the restraint on OMA1. This activates OMA1, which cleaves OPA1 and promotes cytochrome c release. Clearly, OMA1 and OPA1 are not root causes for cancer. Yet many cancer cells rely on this pathway for survival, which can explain why loss of p53 function promotes tumor growth and confers resistance to chemotherapies.

Long-term photoreceptor rescue in two rodent models of retinitis pigmentosa by adeno-associated virus delivery of Stanniocalcin-1.

Retinal degenerations, including age-related macular degeneration and the retinitis pigmentosa family of diseases, are among the leading causes of legal blindness in the United States. We previously found that Stanniocalcin-1 (STC-1) reduced photoreceptor loss in the S334ter-3 and Royal College of Surgeons rat models of retinal degeneration. The results were attributed in part to a reduction in oxidative stress. Herein, we tested the hypothesis that long-term delivery of STC-1 would provide therapeutic rescue in more chronic models of retinal degeneration. To achieve sustained delivery, we produced an adeno-associated virus (AAV) construct to express STC-1 (AAV-STC-1) under the control of a retinal ganglion cell targeting promoter human synapsin 1 (hSYN1). AAV-STC-1 was injected intravitreally into the P23H-1 and S334ter-4 rhodopsin transgenic rats at postnatal day 10. Tissues were collected at postnatal day 120 for confirmation of STC-1 overexpression and histologic and molecular analysis. Electroretinography (ERG) was performed in a cohort of animals at that time. Overexpression of STC-1 resulted in a significant preservation of photoreceptors as assessed by outer nuclear thickness in the P23H-1 (P < 0.05) and the S334ter-4 (P < 0.005) models compared to controls. Additionally, retinal function was significantly improved in the P23H-1 model with overexpressed STC-1 as assessed by ERG analysis (scotopic b-wave P < 0.005 and photopic b-wave P < 0.05). Microarray analysis identified common downstream gene expression changes that occurred in both models. Genes of interest based on their function were selected for validation by quantitative real-time PCR and were significantly increased in the S334ter-4 model.

Individuals with progranulin haploinsufficiency exhibit features of neuronal ceroid lipofuscinosis.

Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous GRN mutations, on the other hand, lead to complete PGRN loss and cause neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease usually seen in children. Given that the predominant clinical and pathological features of FTD and NCL are distinct, it is controversial whether the disease mechanisms associated with complete and partial PGRN loss are similar or distinct. We show that PGRN haploinsufficiency leads to NCL-like features in humans, some occurring before dementia onset. Noninvasive retinal imaging revealed preclinical retinal lipofuscinosis in heterozygous GRN mutation carriers. Increased lipofuscinosis and intracellular NCL-like storage material also occurred in postmortem cortex of heterozygous GRN mutation carriers. Lymphoblasts from heterozygous GRN mutation carriers accumulated prominent NCL-like storage material, which could be rescued by normalizing PGRN expression. Fibroblasts from heterozygous GRN mutation carriers showed impaired lysosomal protease activity. Our findings indicate that progranulin haploinsufficiency caused accumulation of NCL-like storage material and early retinal abnormalities in humans and implicate lysosomal dysfunction as a central disease process in GRN-associated FTD and GRN-associated NCL.

Col4a1 mutations cause progressive retinal neovascular defects and retinopathy.

Mutations in collagen, type IV, alpha 1 (COL4A1), a major component of basement membranes, cause multisystem disorders in humans and mice. In the eye, these include anterior segment dysgenesis, optic nerve hypoplasia and retinal vascular tortuosity. Here we investigate the retinal pathology in mice carrying dominant-negative Col4a1 mutations. To this end, we examined retinas longitudinally in vivo using fluorescein angiography, funduscopy and optical coherence tomography. We assessed retinal function by electroretinography and studied the retinal ultrastructural pathology. Retinal examinations revealed serous chorioretinopathy, retinal hemorrhages, fibrosis or signs of pathogenic angiogenesis with chorioretinal anastomosis in up to approximately 90% of Col4a1 mutant eyes depending on age and the specific mutation. To identify the cell-type responsible for pathogenesis we generated a conditional Col4a1 mutation and determined that primary vascular defects underlie Col4a1-associated retinopathy. We also found focal activation of Müller cells and increased expression of pro-angiogenic factors in retinas from Col4a1(+/Δex41)mice. Together, our findings suggest that patients with COL4A1 and COL4A2 mutations may be at elevated risk of retinal hemorrhages and that retinal examinations may be useful for identifying patients with COL4A1 and COL4A2 mutations who are also at elevated risk of hemorrhagic strokes.

Aging and Vision.

Aging involves defined genetic, biochemical and cellular pathways that regulate lifespan. These pathways are called longevity pathways and they have relevance for many age-related diseases. In the eye, longevity pathways are involved in the major blinding diseases, cataract, glaucoma, age-related macular degeneration (AMD) and diabetic retinopathy. Pharmaceutical targeting of longevity pathways can extend healthy lifespan in laboratory model systems. This offers the possibility of therapeutic interventions to also delay onset or slow the progression of age-related eye diseases. I suggest that retinal degeneration may be viewed as accelerated aging of photoreceptors and that interventions extending healthy lifespan may also slow the pace of photoreceptor loss.

Type IV Collagens and Basement Membrane Diseases: Cell Biology and Pathogenic Mechanisms.

Basement membranes are highly specialized extracellular matrices. Once considered inert scaffolds, basement membranes are now viewed as dynamic and versatile environments that modulate cellular behaviors to regulate tissue development, function, and repair. Increasing evidence suggests that, in addition to providing structural support to neighboring cells, basement membranes serve as reservoirs of growth factors that direct and fine-tune cellular functions. Type IV collagens are a major component of all basement membranes. They evolved along with the earliest multicellular organisms and have been integrated into diverse fundamental biological processes as time and evolution shaped the animal kingdom. The roles of basement membranes in humans are as complex and diverse as their distributions and molecular composition. As a result, basement membrane defects result in multisystem disorders with ambiguous and overlapping boundaries that likely reflect the simultaneous interplay and integration of multiple cellular pathways and processes. Consequently, there will be no single treatment for basement membrane disorders, and therapies are likely to be as varied as the phenotypes. Understanding tissue-specific pathology and the underlying molecular mechanism is the present challenge; personalized medicine will rely upon understanding how a given mutation impacts diverse cellular functions.

Strain-Dependent Anterior Segment Dysgenesis and Progression to Glaucoma in Col4a1 Mutant Mice.

PURPOSE: Mutations in the gene encoding collagen type IV alpha 1 (COL4A1) cause multisystem disorders including anterior segment dysgenesis (ASD) and optic nerve hypoplasia. The penetrance and severity of individual phenotypes depends on genetic context. Here, we tested the effects of a Col4a1 mutation in two different genetic backgrounds to compare how genetic context influences ocular dysgenesis, IOP, and progression to glaucoma. METHODS: Col4a1 mutant mice maintained on a C57BL/6J background were crossed to either 129S6/SvEvTac or CAST/EiJ and the F1 progeny were analyzed by slit-lamp biomicroscopy and optical coherence tomography. We also measured IOPs and compared tissue sections of eyes and optic nerves. RESULTS: We found that the CAST/EiJ inbred strain has a relatively uniform and profound suppression on the effects of Col4a1 mutation and that mutant CASTB6F1 mice were generally only very mildly affected. In contrast, mutant 129B6F1 mice had more variable and severe ASD and IOP dysregulation that were associated with glaucomatous signs including lost or damaged retinal ganglion cell axons and excavation of the optic nerve head. CONCLUSIONS: Ocular defects in Col4a1 mutant mice model ASD and glaucoma that are observed in a subset of patients with COL4A1 mutations. We demonstrate that different inbred strains of mice give graded severities of ASD and we detected elevated IOP and glaucomatous damage in 129B6F1, but not CASTB6F1 mice that carried a Col4a1 mutation. These data demonstrate that genetic context differences are one factor that may contribute to the variable penetrance and severity of ASD and glaucoma in patients with COL4A1 mutations.

In Vivo Visualization of Endoplasmic Reticulum Stress in the Retina Using the ERAI Reporter Mouse.

PURPOSE: Endoplasmic reticulum (ER) stress activates inositol requiring enzyme 1 (IRE1), a key regulator of the unfolded protein response. The ER stress activated indicator (ERAI) transgenic mouse expresses a yellow fluorescent GFP variant (Venus) when IRE1 is activated by ER stress. We tested whether ERAI mice would allow for real-time longitudinal studies of ER stress in living mouse eyes. METHODS: We chemically and genetically induced ER stress, and qualitatively and quantitatively studied the Venus signal by fluorescence ophthalmoscopy. We determined retinal cell types that contribute to the signal by immunohistology, and we performed molecular and biochemical assays using whole retinal lysates to assess activity of the IRE1 pathway. RESULTS: We found qualitative increase in vivo in fluorescence signal at sites of intravitreal tunicamycin injection in ERAI eyes, and quantitative increase in ERAI mice mated to RhoP23H mice expressing ER stress-inducing misfolded rhodopsin protein. As expected, we found that increased Venus signal arose primarily from photoreceptors in RhoP23H/+;ERAI mice. We found increased Xbp1S and XBP1s transcriptional target mRNA levels in RhoP23H/+;ERAI retinas compared to Rho+/+;ERAI retinas, and that Venus signal increased in ERAI retinas as a function of age. CONCLUSIONS: Fluorescence ophthalmoscopy of ERAI mice enables in vivo visualization of retinas undergoing ER stress. ER stress activated indicator mice enable identification of individual retinal cells undergoing ER stress by immunohistochemistry. ER stress activated indicator mice show higher Venus signal at older ages, likely arising from amplification of basal retinal ER stress levels by GFP's inherent stability.

Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.

Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multidomain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans autophosphorylation that further drives homo-oligomerization of its cytosolic kinase/endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase-Inhibiting RNase Attenuators-KIRAs-that allosterically inhibit IRE1α's RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic ß cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate but can itself be controlled with small molecules to reduce cell degeneration.

Dominant optic atrophy, OPA1, and mitochondrial quality control: understanding mitochondrial network dynamics.

Mitochondrial quality control is fundamental to all neurodegenerative diseases, including the most prominent ones, Alzheimer's Disease and Parkinsonism. It is accomplished by mitochondrial network dynamics - continuous fission and fusion of mitochondria. Mitochondrial fission is facilitated by DRP1, while MFN1 and MFN2 on the mitochondrial outer membrane and OPA1 on the mitochondrial inner membrane are essential for mitochondrial fusion. Mitochondrial network dynamics are regulated in highly sophisticated ways by various different posttranslational modifications, such as phosphorylation, ubiquitination, and proteolytic processing of their key-proteins. By this, mitochondria process a wide range of different intracellular and extracellular parameters in order to adapt mitochondrial function to actual energetic and metabolic demands of the host cell, attenuate mitochondrial damage, recycle dysfunctional mitochondria via the mitochondrial autophagy pathway, or arrange for the recycling of the complete host cell by apoptosis. Most of the genes coding for proteins involved in this process have been associated with neurodegenerative diseases. Mutations in one of these genes are associated with a neurodegenerative disease that originally was described to affect retinal ganglion cells only. Since more and more evidence shows that other cell types are affected as well, we would like to discuss the pathology of dominant optic atrophy, which is caused by heterozygous sequence variants in OPA1, in the light of the current view on OPA1 protein function in mitochondrial quality control, in particular on its function in mitochondrial fusion and cytochrome C release. We think OPA1 is a good example to understand the molecular basis for mitochondrial network dynamics.

Down-regulation of OPA1 alters mouse mitochondrial morphology, PTP function, and cardiac adaptation to pressure overload.

AIMS: The optic atrophy 1 (OPA1) protein is an essential protein involved in the fusion of the mitochondrial inner membrane. Despite its high level of expression, the role of OPA1 in the heart is largely unknown. We investigated the role of this protein in Opa1(+/-) mice, having a 50% reduction in OPA1 protein expression in cardiac tissue. METHODS AND RESULTS: In mutant mice, cardiac function assessed by echocardiography was not significantly different from that of the Opa1(+/+). Electron and fluorescence microscopy revealed altered morphology of the Opa1(+/-) mice mitochondrial network; unexpectedly, mitochondria were larger with the presence of clusters of fused mitochondria and altered cristae. In permeabilized mutant ventricular fibres, mitochondrial functional properties were maintained, but direct energy channelling between mitochondria and myofilaments was weakened. Importantly, the mitochondrial permeability transition pore (PTP) opening in isolated permeabilized cardiomyocytes and in isolated mitochondria was significantly less sensitive to mitochondrial calcium accumulation. Finally, 6 weeks after transversal aortic constriction, Opa1(+/-) hearts demonstrated hypertrophy almost two-fold higher (P< 0.01) than in wild-type mice with altered ejection fraction (decrease in 43 vs. 22% in Opa1(+/+) mice, P< 0.05). CONCLUSIONS: These results suggest that, in adult cardiomyocytes, OPA1 plays an important role in mitochondrial morphology and PTP functioning. These properties may be critical for cardiac function under conditions of chronic pressure overload.

A clinically complex form of dominant optic atrophy (OPA8) maps on chromosome 16.

Dominant optic atrophy (DOA) is genetically heterogeneous and pathogenic mutations have been identified in the OPA1 and OPA3 genes, both encoding for mitochondrial proteins. We characterized clinical and laboratory features in a large OPA1-negative family with complicated DOA. Search for mitochondrial dysfunction was performed by studying muscle biopsies, fibroblasts, platelets and magnetic resonance (MR) spectroscopy. Genetic investigations included mitochondrial DNA (mtDNA) analysis, linkage analysis, copy number variation (CNV) analysis and candidate gene screening. Optic neuropathy was undistinguishable from that in OPA1-DOA and frequently associated with late-onset sensorineural hearing loss, increases of central conduction times at somato-sensory evoked potentials and various cardiac abnormalities. Serum lactic acid after exercise, platelet respiratory complex activities, adenosine triphosphate (ATP) content in fibroblasts and muscle phosphorus MR spectroscopy all failed to reveal a mitochondrial dysfunction. However, muscle biopsies and their mtDNA analysis showed increased mitochondrial biogenesis. Furthermore, patient's fibroblasts grown in the galactose medium were unable to increase ATP content compared with controls, and exhibited abnormally high rate of fusion activity. Genome-wide linkage revealed a locus on chromosome 16q21-q22 with a maximum two-point LOD score of 8.84 for the marker D16S752 and a non-recombinant interval of ∼ 6.96 cM. Genomic screening of 45 genes in this interval including several likely candidate genes (CALB2, CYB5B, TK2, DHODH, PLEKHG4) revealed no mutation. Moreover, we excluded the presence of CNVs using array-based comparative genome hybridization. The identification of a new OPA locus (OPA8) in this pedigree demonstrates further genetic heterogeneity in DOA, and our results indicate that the pathogenesis may still involve mitochondria.

Solving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with autosomal dominant optic atrophy.

BACKGROUND: Up to the 1950s, there was an ongoing debate about the diversity of hereditary optic neuropathies, in particular as to whether all inherited optic atrophies can be ascribed to Leber's hereditary optic neuropathy (LHON) or represent different disease entities. In 1954 W. Jaeger published a detailed clinical and genealogical investigation of a large family with explicit autosomal dominant segregation of optic atrophy thus proving the existence of a discrete disease different from LHON, which is nowadays known as autosomal dominant optic atrophy (ADOA). Since the year 2000 ADOA is associated with genomic mutations in the OPA1 gene, which codes for a protein that is imported into mitochondria where it is required for mitochondrial fusion. Interestingly enough, the underlying mutation in this family has not been identified since then. RESULTS: We have reinvestigated this family with the aim to identify the mutation and to further clarify the underlying pathomechanism. Patients showed a classical non-syndromic ADOA. The long term deterioration in vision in the two teenagers examined 50 years later is of particular note 5/20 to 6/120. Multiplex ligation probe amplification revealed a duplication of the OPA1 exons 7-9 which was confirmed by long distance PCR and cDNA analysis, resulting in an in-frame duplication of 102 amino acids. Segregation was verified in 53 available members of the updated pedigree and a penetrance of 88% was calculated. Fibroblast cultures from skin biopsies were established to assess the mitochondrial network integrity and to qualitatively and quantitatively study the consequences of the mutation on transcript and protein level. Fibroblast cultures demonstrated a fragmented mitochondrial network. Processing of the OPA1 protein was altered. There was no correlation of the OPA1 transcript levels and the OPA1 protein levels in the fibroblasts. Intriguingly an overall decrease of mitochondrial proteins was observed in patients' fibroblasts, while the OPA1 transcript levels were elevated. CONCLUSIONS: The thorough study of this family provides a detailed clinical picture accompanied by a molecular investigation of patients' fibroblasts. Our data show a classic OPA1-associated non-syndromic ADOA segregating in this family. Cell biological findings suggest that OPA1 is regulated by post-translational mechanisms and we would like to hypothesize that loss of OPA1 function might lead to impaired mitochondrial quality control. With the clinical, genetic and cell biological characterisation of a family described already more than 50 years ago, we span more than half a century of research in optic neuropathies.

Electrophysiological and histologic assessment of retinal ganglion cell fate in a mouse model for OPA1-associated autosomal dominant optic atrophy.

PURPOSE: The main disease features of autosomal dominant optic atrophy (ADOA) are a bilateral reduction of visual acuity, cecocentral scotoma, and frequently tritanopia, which have been ascribed to a progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. The main disease-causing gene is OPA1. Here, we examine a mouse carrying a pathogenic mutation in Opa1 by electrophysiological measurements and assess the fate of RGCs. METHODS: Two-year-old animals underwent a full examination by electroretinography (ERG) and visually evoked potential (VEP) measurements to assess the function of the outer and inner retina and the optic nerve. Retrograde Fluorogold labeling was performed to determine the number of surviving RGCs and to assess axonal transport by neurofilament counterstaining. Phagocytosis-dependent labeled microglial cells were identified by an Iba-1 staining. RESULTS: ERG responses were normal in aged Opa1 mice. VEP measurements revealed significantly reduced amplitudes but no change in the latencies in contrast to extended latencies found in glaucoma. Retrograde labeling of RGCs showed a significant reduction in the number of RGCs in Opa1 mice. Long-term experiments revealed the presence of microglial cells with ingested fluorescent dye. CONCLUSIONS: This is the first electrophysiological demonstration of a visual function deficit in aged Opa1 mice. VEP measurements and retrograde labeling experiments show that the number of RGCs is reduced whereas the remaining RGCs and axons function normally. Taken together, these findings support an ascending progress of degeneration from the soma toward the axon.

Subtle neurological and metabolic abnormalities in an Opa1 mouse model of autosomal dominant optic atrophy.

The ubiquitously expressed gene OPA1 is the main disease causing gene for autosomal dominant optic atrophy (ADOA). These patients present with bilateral reduction in visual acuity, central visual field defects and impaired color vision, secondary to the progressive loss of retinal ganglion cells (RGCs) and subsequent degeneration of the optic nerve. Up to now, it is not clear why a mutation in a ubiquitously expressed gene affects only RGCs and the optic nerve. Twenty-two-month-old Opa1 animals underwent a full examination following the Shirpa protocol. Weight, food intake and life span were monitored. Rotarod treadmill experiments were performed to assess neuromuscular function. Limb skeletal muscle was evaluated morphologically, mitochondrial cytochrome c oxidase (COX) activity was studied histochemically and mtDNA integrity was determined by long-range PCR. The Shirpa test showed that 33% of the Opa1 mice suffered from tremor and 52% of the Opa1 animals showed an abnormal clutching reflex. Control animals performed well in the accelerating Rotarod treadmill experiment whereas the Opa1 mice performed significantly worse. Skeletal muscle fibers were morphologically normal, had normal COX activity and showed no evidence of secondary mtDNA damage in contrast to patients with syndromic ADOA. We also found a highly significant difference in body weight. Our results demonstrate that OPA1 mutations affect not only RGCs but also other tissues and cell types, though to a lesser extent. In particular we found deficits in both neuromuscular and metabolic function. We therefore want to encourage clinicians to be vigilant about to extra-ocular manifestations in ADOA patients.

Identification of genes that are linked with optineurin expression using a combined RNAi--microarray approach.

Mutations in the optineurin gene are associated with open-angle glaucoma. Its gene product is a 74 kDa protein implicated in several cellular pathways. Although a range of interacting partners of optineurin have been identified, its physiological and pathophysiological role remains unclear. To understand comprehensive molecular mechanisms by which optineurin mediates, we identified genome-wide molecular changes upon silencing optineurin in HeLa cells by using microarray technology. A series of differentially expressed genes due to reduced expression of optineurin was identified. Network analyses showed that most of the functional categories of identified genes are associated with cellular function and maintenance as well as cellular assembly and organization. From these networks 22 genes were selected for confirmation by quantitative real-time PCR (Q-RT-PCR). To eliminate false-positive results due to off-target effects, a second siRNA was used to transfect HeLa cells and candidate genes were re-analyzed in these samples applying Q-RT-PCR. Several genes turned out to be differentially expressed in both siRNA experiments and changes in expression were confirmed on protein level. Coupling RNAi knockdown with microarray and Q-RT-PCR analyses provided several candidate genes that are linked with optineurin expression and confirms the assumption that optineurin is involved in trafficking processes and cellular morphology.