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Subtle neurological and metabolic abnormalities in an Opa1 mouse model of autosomal dominant optic atrophy
Experimental Neurology 220 (2009) 404–409
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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Brief Communication
Subtle neurological and metabolic abnormalities in an Opa1 mouse model of autosomal dominant optic atrophy Marcel V. Alavi a,⁎,1, Nico Fuhrmann a,1, Huu Phuc Nguyen b, Patrick Yu-Wai-Man c,d, Peter Heiduschka e, Patrick F. Chinnery c, Bernd Wissinger a a
Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Germany Department of Medical Genetics, University of Tuebingen, Germany Mitochondrial Research Group, Institute for Ageing and Health, The Medical School, Newcastle University, UK d Department of Ophthalmology, Royal Victoria Infirmary, Newcastle upon Tyne, UK e Experimental Vitreoretinal Surgery, Centre for Ophthalmology, University of Tuebingen, Germany b c
a r t i c l e
i n f o
Article history: Received 26 July 2009 Revised 9 September 2009 Accepted 28 September 2009 Available online 6 October 2009 Keywords: Opa1 ADOA Mouse mtDNA Rotarod Accelerod Neuromuscular abnormalities Subclinical Life expectancy SHIRPA Tremor Limb grasping
a b s t r a c t 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. © 2009 Elsevier Inc. All rights reserved.
Introduction With an estimated prevalence of up to 1:12,000 in Denmark (Kivlin et al., 1983; Kjer et al., 1996) autosomal dominant optic atrophy (OMIM 165500, ADOA) is one of the most common hereditary optic neuropathies. It is characterized clinically by a variable loss of visual acuity, central field defect, reduced color discrimination and pallor of the optic nerve head (Jaeger, 1966; Lorenz, 1994). There is considerable intrafamilial and interfamilial variability in progression and severity of the visual defects ranging from functionally asymptomatic carriers to legally blind patients (Caldwell et al., 1971; Kline and Glaser, 1979; Roggeveen et al., 1985). Histopathological
⁎ Corresponding author. Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Roentgenweg 11, D72076 Tuebingen, Germany. Fax: +49 0 7071 29 5725. E-mail address: [email protected] (M.V. Alavi). 1 Contributed equally. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.09.026
examinations of affected donor eyes confirm substantial loss of retinal ganglion cells (RGCs) with secondary atrophy of the optic nerve (Barbet et al., 2005; Kerrison et al., 1999). Although several loci have been described, indicating genetic heterogeneity in ADOA (Barbet et al., 2005; Kerrison et al., 1999; Reynier et al., 2004), mutations in optic atrophy gene 1 (OPA1) constitute the main cause for ADOA (Alexander et al., 2000; Delettre et al., 2000). The OPA1 protein is a nuclear encoded, dynamin related GTPase, which is imported into mitochondria and anchored in the inner membrane of the organelle (Olichon et al., 2002). Together with mitofusin 1 and mitofusin 2 it plays a major role in mitochondrial fusion and therefore is important for the maintenance of the mitochondrial network and morphology (Cipolat et al., 2004). Furthermore OPA1 is linked to mitochondrial cristae remodeling in apoptosis (Cipolat et al., 2006; Frezza et al., 2006; Olichon et al., 2003). However, the exact biochemical role of OPA1 is still under investigation. The OPA1 gene is expressed ubiquitously (Alexander et al., 2000; Bette et al., 2005) with the highest levels in retina, brain, testis, heart,
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and muscle (Alexander et al., 2000). The essential and fundamental biological role of OPA1 became evident with the examination of the embryogenesis of homozygous mutant mice, which are not viable and die in utero (Alavi et al., 2007; Davies et al., 2007). To date 204 different pathogenic mutations in OPA1 have been described that are all associated with optic atrophy (Ferre et al., 2005). In this respect it is particularly interesting that heterozygous mutations in OPA1 apparently affect only the RGCs. Haploinsufficiency is believed to be a major pathomechanism in OPA1 gene related non-syndromic ADOA (Fuhrmann et al., 2009; Marchbank et al., 2002), but there are case reports of syndromic forms of ADOA that include extra-ocular neurological features (sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia, mitochondrial myopathy) that are associated with multiple mitochondrial DNA (mtDNA) deletions implicating a possible role in mtDNA replication (Amati-Bonneau et al., 2008; Hudson et al., 2008; Zeviani, 2008). In this study we have examined an Opa1 mutant mouse model for ADOA with special emphasis on subtle neurological abnormalities. Previously, it has been shown that this Opa1 mouse shows a slow degeneration of the optic nerve with a progressive loss of RGCs (Alavi et al., 2007). On histology, prominent loss of RGCs was observed in animals that were at least 17 months of age; on the other hand there is variability in the disease manifestation, since a majority of the aged animals showed apparently no signs of RGC loss (Alavi et al., 2007). Recently we presented an electrophysiological characterization of this Opa1 mouse mutant, where we demonstrate that the RGCs are in fact primarily affected and that the Opa1 mutation impairs directly the survival of RGCs (Heiduschka et al., 2009). In this study, we provide evidence that Opa1 mutant mice develop neuromuscular symptoms at late ages. In line with previous results, these impairments show variable severity. Since the Opa1 mouse strain is not a congenic inbred this phenotypic variability points to the presence of genetic modifiers. These neuromuscular symptoms are not associated with altered muscle fiber morphology, COX activity or the presence of multiple mtDNA deletions. Although life expectancy was not shortened in our OPA1 mice, our observations also point to possible subclinical alteration in metabolism because Opa1 mice showed reduced body fat compared with controls.
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Accelerod test To determine fore and hind limb motor coordination and balance, a previously described accelerod test (Nguyen et al., 2006) was adapted for our mice. Briefly, this apparatus consisted of a base platform and a rotating rod with a non-skid surface. When operated in the acceleration modus, the rotor would accelerate from 4 to 40 rpm in a period of 5 min. Before testing, mice were trained four times a day for 2 min on three consecutive days. By this time, a steady baseline level of performance was attained. Opa1 mice (n = 13; 7 ♂ + 6 ♀) and controls (n = 6; 3 ♂ + 3 ♀) were tested at an age of 22 months starting with 4 rpm and subsequently accelerating after 30 s gradually up to 40 rpm in 5 min. The time spent on the rod before falling off was recorded and four independent experiments were averaged. To reveal the substantial variation of performance each result of the 4 single experiments has been included in the box-plots. mtDNA deletions The integrity of the mtDNA has been assessed as described previously (Tyynismaa et al., 2005) in DNA extracted (High Pure PCR Template Preparation Kit, Roche Diagnostics, Mannheim, Germany) from homogenate muscle tissue of 22-month-old mice. Briefly, long distance PCR (TaKaRa LA Taq, TaKaRa, Shiga, Japan) applying oligos: mito_2473-2505_for: 5′-GGT TCG TTT GTT CAA CGA TTA AAG TCC TAC GTG-3′ and mito_1953-1924_rev: 5′-GAG GTG ATG TTT TTG GTA AAC AGG CGG GGT-3′ was used to amplify a 16 kb fragment of mouse mtDNA in a 2 step cycling reaction (98°C for 10 s and 68°C for 12 min; 35 cycles). Histochemistry Observed differences between Opa1 mice and controls were considered significant with a p value b 0.05 and highly significant with a p value b 0.005 in an unpaired t-test. If not stated differently, all numbers are mean values ± standard deviation (SD). Kaplan–Meier plots were calculated for each group using SigmaPlot 2001 (SPSS, Chicago, Illinois, USA). Box plots show median, 25th and 75th percentiles as boxes, 10th and 90th percentiles as whiskers and outliners as dots. Statistics
Experimental methods Animals Mice were kept in a 12 h light (10 lx)/12 h dark cycle with food and water available ad libitum in full-barrier facilities free of specific pathogens. Mouse breeding and all experimental procedures were done according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by legal authorities. The Opa1enu/+ strain (abbreviated Opa1 mouse) has been initially described by our group elsewhere (Alavi et al., 2007). All examined mice were at least 22 months old.
The gastrocnemius muscle was harvested from the leg area of the following 22-month-old mice: Opa1+/+ (n = 5), non-affected Opa1+/- (n = 4) i.e. normal performance on the accelerod test, and affected Opa1+/- (n = 3), and immediately frozen in a melting isopentane bath (-150°C). Serial 20 μm muscle sections were cut and mounted onto glass slides using a Microm™ HM560 cryostat (Thermo Fisher, Germany), and these were then stained using established protocols for cytochrome c oxidase (COX), succinate dehydrogenase (SDH) and dual COX-SDH (Johnson and Barron, 1996, Taylor et al., 2004). Myofibrillar ATPase staining was also performed at a preincubation pH of 4.3 to further define the pattern of oxidative Type I (Dark) and glycolytic Type II (Light grey) fibers in these muscle sections (Johnson and Barron, 1996).
Primary phenotyping Results Thirty-nine aged Opa1 mice (24 ♂ + 15 ♀) and 15 age-matched controls (9 ♂ + 6 ♀) were first examined according to the modified SHIRPA protocol (European Mouse Phenotyping Resource of Standardised Screens; EMPReSS; http://empress.har.mrc.ac.uk/), which assesses mouse behavior in a viewing jar and above an arena. This screening was performed by two independent observers on 2 days. Furthermore, body weight and 24 h food intake were recorded for 32 Opa1 mice (20 ♂ + 12 ♀) and 10 (6 ♂ + 4 ♀) controls.
Primary SHIRPA screen We used the primary SHIRPA screen to assess the muscle and motor neuron function of a group of 39 22-month-old Opa1 mice and compared these results with those of 15 wild-type (WT) littermates (control). The Opa1 group included 15 females and 24 males (ratio ♀/♂ = 0.625) and hence was comparable to the control
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group with 6 females and 9 males (ratio ♀/♂ = 0.667). There were no significant differences for the following parameters recorded in a viewing jar: body position, palpebral closure, coat appearance, whiskers, lacrimation, defecation and skin color. One parameter differed significantly: 33% of the Opa1 mice showed an obvious resting tremor of the upper limbs versus none in the control group. With 2 ♀ vs. 11 ♂ there was also an imbalance toward males being more prone to the development of a tremor. The parameters of positional passivity, evidence of biting and vocalization that have been recorded above the arena, did not differ significantly, but 52% of the Opa1 mice showed an abnormal clasping of the hind limbs when suspended by their tail. There was no sex difference in terms of clasping, and none of the controls exhibited this behavior. Body weight and life span We found a highly significant difference in body weight. Body weights were determined for Opa1 mice with an average age of 635 days (SD = 30 days) and compared with controls with a mean age of 640 days (SD = 22 days; Fig. 1A). Under regular animal housing conditions, all animals in the control group were morbidly obese (mean: 48.9 g; SD = 7.2 g), while the Opa1 mice showed no signs of obesity (34.9 g; SD = 4.1 g; Figs. 1B and 2A). This was highly significant (p value = 1.9⁎10-9). Sex had only little effect on the body weight and differed within 1.6 g for both groups (not shown). There was no significant difference in food intake (Fig. 1C). A 24 h study revealed an average 24 h dry food intake of 4.7 g (SD = 0.5 g) for the controls and 4.4 g (SD = 0.9 g) for Opa1 mice (p value = 0.3597). Post mortem examinations revealed that the Opa1 animals had substantially less body fat compared to controls (Fig. 2A). Life expectancy was not reduced in the Opa1 group (mean life time: 765 days; SE = 25 days) when compared to the control group (758 days; SE = 19 days; Kaplan–Meier log rank test: p value = 0.42;
Fig. 1D). It did also not vary from data of a previous thorough study on the life expectancy of 746 C57Bl6 mice (Rowlatt et al., 1976). Accelerod test Motor coordination and balance capabilities of mice were measured using an accelerating Rotarod treadmill (accelerod) in an age-matched (mean age = 661 days; SD = 20 days) group of Opa1 animals and controls (mean age = 667 days; SD = 25). All control animals performed well in this experiment and acquired the task within the given time of training. In the Opa1 group some mice showed a clear disability to manage the task, while the remaining Opa1 animals' performance was indistinguishable from the control group. This is also reflected in the mean running time of the individual animals (Fig. 2B). While all controls taken together had a mean running time (mrt) of 88.7 s (SD = 26.4 s), Opa1 mice (mrt = 70.2 s; SD = 28.4 s) performed significantly worse (p value = 0.0352) than the controls (Fig. 2C; Control and Opa1 oa). If one would like to speculate, one could combine all Opa1 mice that had difficulties to manage the task (Opa1 aff.; mrt = 54.6 s; SD = 19.6 s) and compare it with the remaining Opa1 animals (Opa1 not aff.; mrt = 88.4 s; SD = 26.3 s; p value = 0.0001) or controls (p value = 0.0002; Fig. 2C). Noteworthy 3 out of 7 Opa1 animals which performed significantly worse in the accelerod experiments presented with tremor, whereas none of the well performing Opa1 animals had a tremor. The abnormal clutching reflex could be observed in 52% of the Opa1 animals independently of their performance in the accelerod test. Muscle morphology, COX activity and mtDNA integrity The Opa1 mouse has been considered as a model for nonsyndromic ADOA (Alavi et al., 2007), while certain forms of syndromic ADOA have been associated with mtDNA deletions (Amati-Bonneau et
Fig. 1. Opa1 mice show significantly less obesity than controls. Life expectancy is not altered for Opa1 mice. (A) Mean age of the mice ± SD at examination for weight and food intake. (B) Box plot of body weight, dashed line indicates the mean weight. (C) Mean food intake in 24 h of the mice ± SD. (D) Kaplan–Meier plot of the life expectancy for Opa1 and control mice (relative risk = 1.37; standard error 0.531; p value = 0.42).
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Fig. 2. Opa1 mice performed significantly worse in accelerod experiments. (A) Control mouse (left) and Opa1 mouse (right) on a treadmill. (B) Mean running time of 4 independent experiments for each single animal (error bars = SD). Last lane gives the mean running time of all animals of the according group. Opa1 mice performed significantly worse than controls. (C) Box plot of the accelerod performance for controls and Opa1 mice (Opa1 oa) depicting the single results of each run. Though speculative, if one combines all Opa1 mice that performed similar or better than the average (Opa1 not aff.; mouse 1 to 6 in B) and Opa1 mice that performed worse than the average (Opa1 aff.; mouse 7 to 13 in B), the Opa1 not aff. group performed similar to controls while the Opa1 aff. group performed highly significantly worse.
al., 2008; Hudson et al., 2008; Zeviani, 2008). Since our initial findings suggested subtle neuromuscular abnormalities, we analyzed the integrity of mtDNA in homogenate muscle samples derived from 22-month-old Opa1 mice and age-matched controls. We found minor mtDNA deletion bands in the majority of the analyzed muscle samples, from both Opa1 and control animals (Fig. 3A). However, all samples revealed a substantial content of intact mtDNA, which was not the case for homogenate muscle mtDNA derived from 18-monthold “deletor” mice, a model for progressive external ophthalmoplegia, which is caused by mutations in the mtDNA helicase twinkle that accumulates mtDNA deletions over time (Tyynismaa et al., 2005) (Fig. 3A, line 15). Histochemistry revealed normal muscle fiber morphology and no ragged red fibers (RRFs) or cytochrome c oxidase (COX) deficient fibers. Myofibrillar ATPase staining showed a preponderance of Type II fibers (90%–95%) with no observable pathological grouping of Type I fibers in mice of all 3 groups (Figs. 3B and C). Discussion The OPA1 gene is expressed ubiquitously (Alexander et al., 2000; Bette et al., 2005) and has been associated with fundamental processes such as mitochondrial fusion, apoptotic cristae remodeling and cytochrome c release (Cipolat et al., 2004, 2006; Frezza et al., 2006; Olichon et al., 2007). OPA1 homologs are known in yeast and all higher eukaryotes (Hales and Fuller, 1997). The importance of OPA1 is furthermore emphasized by the fact that mice with homozygous mutations in Opa1 are not viable and die during embryogenesis (Alavi
et al., 2007). In contrast the vast majority of heterozygous OPA1 mutations are associated with non-syndromic ADOA in humans (Ferre et al., 2005), that affect only the survival of RGCs. This led to the formulation of a fundamental question: What makes retinal ganglion cells unique that they are the only cells which are affected by OPA1 mutations? (Yu Wai Man et al., 2005). It has been hypothesized that the retina as highly oxygen consuming tissue and the optic nerve with its initial non-myelinated part might be especially prone to mitochondrial disturbances which would explain a mainly restricted ocular phenotype. However, can we exclude that OPA1 mutations do not affect other cell types or organs at subclinical levels? In other words: Why should heterozygous mutations in such a fundamental important gene like OPA1 impair solely the function of a single cell type? To address this question, we re-examined aged 22-month-old Opa1 mice (Alavi et al., 2007) for subtle phenotypic abnormalities. Using the primary SHIRPA screen, we found that about half of the Opa1 mice showed an abnormal clutching of their hind limbs. This indicates defects in brain, spinal cord or dorsal root ganglia (Klein et al., 1994; Mangiarini et al., 1996). One third of the Opa1 mice presented with a resting tremor of their upper limbs, suggesting defects of the brain stem or the cerebellum (Friedman et al., 2007). The Opa1 mice performed significantly worse in the accelerod experiment compared to controls, which points to neuromuscular coordination difficulties of the Opa1 mice (Nguyen et al., 2006). In summary, our experiments suggest that the subtle neuromuscular abnormalities observed in the mutant mice manifest relatively late in age and these are likely secondary to involvement of the peripheral
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Fig. 3. (A) Long distance PCR of muscle DNA from Opa1 mice and controls both demonstrated minor mtDNA deletions bands with the presence of a strong undeleted band representing the 16 kb amplicon. (B) ATPase staining with a pre-incubation pH of 4.3 showing a greater proportion of Type II fibers (light gray) compared to Type I fibers (dark), which is normal for mouse gastrocnemius muscle. There was no pathological grouping of Type I fibers. (C) Absence of COX-deficient fibers in muscle sections following dual COX–SDH histochemistry. The darker staining fibers are Type I due to their higher mitochondrial oxidative capacity compared to Type II fibers. These are representative slides from an affected 22-month-old Opa1 mouse but a similar staining pattern was observed in all mice studied. Bar = 200 μm.
and/or central nervous system. However we were not able to correlate the loss of retinal ganglion cells to the performance in the accelerod experiment, in other words a mouse with apparently advanced RGC loss performed very well in the accelerod, while a mouse with minor RGC loss performed worse. See Heiduschka et al. 2009 for a full description of the ocular phenotype. Recent publications demonstrate the accumulation of mtDNA deletions in patients with syndromic forms of OPA1-associated optic atrophy that suffer from severe additional neurological symptoms like sensorineural deafness, ataxia, axonal sensory-motor polyneuropa-
thy, chronic progressive external ophthalmoplegia and mitochondrial myopathy (Amati-Bonneau et al., 2008; Ferraris et al., 2008; Hudson et al., 2008). However histological analyses of muscle sections from Opa1 mice excluded a primary myopathic process and we did not find any evidence for the presence of significant amounts of deleted mtDNA molecules in our muscle samples. The detected minor deletion bands were comparable between Opa1 mice and controls and might originate from normal ageing processes rather than represent a pathologic finding (Taylor et al., 2004). This finding would suggest that our Opa1 mouse is a model for non-syndromic ADOA rather than one for syndromic ADOA. In this context it is of particular importance that the Opa1 mouse does not suffer from any hearing impairment (Alavi et al., 2007). There was also no evidence of COX-deficient fibers and with a minimum sampling of 1000 muscle fibers for each mouse, if COX-deficient fibers were missed, these are likely be present at only low levels (b0.5%) and therefore of no pathological significance. Type II fibers constitute N90% of all fibers in mouse gastrocnemius muscle (Hayasaki et al., 2001; Manttari and Jarvilehto, 2005) and based upon myofibrillar ATPase staining, we observed a similar distribution in our Opa1 mice, irrespective of mutational or neuromuscular status. Importantly, angulated fibers were absent and there was no evidence of pathological grouping of oxidative Type I fibers precluding a denervation process affecting the peripheral nerves. The normal fiber architecture and absence of inflammatory cells on histology also excluded the possibility of an underlying inflammatory or degenerative myopathic process. Therefore we would like to postulate that the pathology is central in origin because we can exclude a myopathy and a peripheral neuropathy, and although we have no data at this stage we would like to speculate about a possible cerebellar origin. The difference in body weight of Opa1 mice compared to the obese controls might indicate an altered metabolism in aged mutant mice, supported by the reduced body fat content in the mutant mice. Although speculative, altered insulin secretion might lead to less accumulation of body fat over time, given the recent findings that the Opa1 protein is involved in mitochondrial maintenance in pancreatic beta cells (Twig et al., 2008). Future experiments are required to investigate metabolic function in Opa1 mice in more details. Only about half of aged animals showed these phenotypic abnormalities. Moreover, these additional phenotypic features do not appear to be linked with the actual severity of RGC loss and hence level of visual impairment. Since the Opa1 mouse strain is not congenic inbred this phenotypic variability points to the presence of genetic modifiers which is in line with the observed strong ocular phenotypic variability in humans (Fuhrmann et al., 2009). Outcross to different genetic backgrounds might help to identify new modifier genes in the future. Taken together, we have shown that RGCs are not the only cell types which are affected by Opa1 mutations in an Opa1 mouse model for ADOA. Moreover, our findings indicate that ADOA with additional, though subclinical extra-ocular manifestations is not necessarily linked with the presence of multiple mtDNA deletions, highlighting the need to further elucidate the other roles played by the OPA1 protein in normal mitochondrial function. Therefore this study complements a recent report on the absence of mtDNA defects in atrophic optic nerves of a second Opa1 mouse model (Yu-Wai-Man et al., 2009). Recently, a subclinical neuromuscular phenotype in an ADOA family without any mtDNA deletions has been reported (Spinazzi et al., 2008), which is in line with our findings. Therefore, we consider it possible that mutations in OPA1 can cause additional subtle neuromuscular impairments even in apparently non-syndromic ADOA patients, because of the crucial role of the ubiquitously expressed OPA1 protein in mitochondrial biogenesis and maintenance, and its involvement in fundamental cellular functions. We therefore want to encourage clinicians to be more attentive to extraocular manifestations in patients with optic atrophy.
Brief Communication Conflict of interest statement The authors wish to declare no competing interest.
Acknowledgments The authors are deeply grateful to Henna Tyynismaa (Neurology, University of Helsinki, Finland) who kindly shared her knowledge and her DNA reference sample of the mutator mouse. We also thank Elke Maier (Anatomy, University Clinics, Tuebingen, Germany) for giving excellent technical advice and providing Isopentane. We are indebted to Klaus Wichmann and Hjörvar Pétursson for help with statistical analysis. PYWM is an MRC Clinical Research Fellow and PFC is a Wellcome Trust Senior Fellow in Clinical Science. References Alavi, M.V., Bette, S., Schimpf, S., Schuettauf, F., Schraermeyer, U., Wehrl, H.F., Ruttiger, L., Beck, S.C., Tonagel, F., Pichler, B.J., Knipper, M., Peters, T., Laufs, J., Wissinger, B., 2007. A splice site mutation in the murine Opa1 gene features pathology of autosomal dominant optic atrophy. Brain 130, 1029–1042. 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Experimental Neurology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y e x n r
Brief Communication
Subtle neurological and metabolic abnormalities in an Opa1 mouse model of autosomal dominant optic atrophy Marcel V. Alavi a,⁎,1, Nico Fuhrmann a,1, Huu Phuc Nguyen b, Patrick Yu-Wai-Man c,d, Peter Heiduschka e, Patrick F. Chinnery c, Bernd Wissinger a a
Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Germany Department of Medical Genetics, University of Tuebingen, Germany Mitochondrial Research Group, Institute for Ageing and Health, The Medical School, Newcastle University, UK d Department of Ophthalmology, Royal Victoria Infirmary, Newcastle upon Tyne, UK e Experimental Vitreoretinal Surgery, Centre for Ophthalmology, University of Tuebingen, Germany b c
a r t i c l e
i n f o
Article history: Received 26 July 2009 Revised 9 September 2009 Accepted 28 September 2009 Available online 6 October 2009 Keywords: Opa1 ADOA Mouse mtDNA Rotarod Accelerod Neuromuscular abnormalities Subclinical Life expectancy SHIRPA Tremor Limb grasping
a b s t r a c t 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. © 2009 Elsevier Inc. All rights reserved.
Introduction With an estimated prevalence of up to 1:12,000 in Denmark (Kivlin et al., 1983; Kjer et al., 1996) autosomal dominant optic atrophy (OMIM 165500, ADOA) is one of the most common hereditary optic neuropathies. It is characterized clinically by a variable loss of visual acuity, central field defect, reduced color discrimination and pallor of the optic nerve head (Jaeger, 1966; Lorenz, 1994). There is considerable intrafamilial and interfamilial variability in progression and severity of the visual defects ranging from functionally asymptomatic carriers to legally blind patients (Caldwell et al., 1971; Kline and Glaser, 1979; Roggeveen et al., 1985). Histopathological
⁎ Corresponding author. Molecular Genetics Laboratory, Institute for Ophthalmic Research, Centre for Ophthalmology, University of Tuebingen, Roentgenweg 11, D72076 Tuebingen, Germany. Fax: +49 0 7071 29 5725. E-mail address: [email protected] (M.V. Alavi). 1 Contributed equally. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.09.026
examinations of affected donor eyes confirm substantial loss of retinal ganglion cells (RGCs) with secondary atrophy of the optic nerve (Barbet et al., 2005; Kerrison et al., 1999). Although several loci have been described, indicating genetic heterogeneity in ADOA (Barbet et al., 2005; Kerrison et al., 1999; Reynier et al., 2004), mutations in optic atrophy gene 1 (OPA1) constitute the main cause for ADOA (Alexander et al., 2000; Delettre et al., 2000). The OPA1 protein is a nuclear encoded, dynamin related GTPase, which is imported into mitochondria and anchored in the inner membrane of the organelle (Olichon et al., 2002). Together with mitofusin 1 and mitofusin 2 it plays a major role in mitochondrial fusion and therefore is important for the maintenance of the mitochondrial network and morphology (Cipolat et al., 2004). Furthermore OPA1 is linked to mitochondrial cristae remodeling in apoptosis (Cipolat et al., 2006; Frezza et al., 2006; Olichon et al., 2003). However, the exact biochemical role of OPA1 is still under investigation. The OPA1 gene is expressed ubiquitously (Alexander et al., 2000; Bette et al., 2005) with the highest levels in retina, brain, testis, heart,
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and muscle (Alexander et al., 2000). The essential and fundamental biological role of OPA1 became evident with the examination of the embryogenesis of homozygous mutant mice, which are not viable and die in utero (Alavi et al., 2007; Davies et al., 2007). To date 204 different pathogenic mutations in OPA1 have been described that are all associated with optic atrophy (Ferre et al., 2005). In this respect it is particularly interesting that heterozygous mutations in OPA1 apparently affect only the RGCs. Haploinsufficiency is believed to be a major pathomechanism in OPA1 gene related non-syndromic ADOA (Fuhrmann et al., 2009; Marchbank et al., 2002), but there are case reports of syndromic forms of ADOA that include extra-ocular neurological features (sensorineural deafness, ataxia, axonal sensory-motor polyneuropathy, chronic progressive external ophthalmoplegia, mitochondrial myopathy) that are associated with multiple mitochondrial DNA (mtDNA) deletions implicating a possible role in mtDNA replication (Amati-Bonneau et al., 2008; Hudson et al., 2008; Zeviani, 2008). In this study we have examined an Opa1 mutant mouse model for ADOA with special emphasis on subtle neurological abnormalities. Previously, it has been shown that this Opa1 mouse shows a slow degeneration of the optic nerve with a progressive loss of RGCs (Alavi et al., 2007). On histology, prominent loss of RGCs was observed in animals that were at least 17 months of age; on the other hand there is variability in the disease manifestation, since a majority of the aged animals showed apparently no signs of RGC loss (Alavi et al., 2007). Recently we presented an electrophysiological characterization of this Opa1 mouse mutant, where we demonstrate that the RGCs are in fact primarily affected and that the Opa1 mutation impairs directly the survival of RGCs (Heiduschka et al., 2009). In this study, we provide evidence that Opa1 mutant mice develop neuromuscular symptoms at late ages. In line with previous results, these impairments show variable severity. Since the Opa1 mouse strain is not a congenic inbred this phenotypic variability points to the presence of genetic modifiers. These neuromuscular symptoms are not associated with altered muscle fiber morphology, COX activity or the presence of multiple mtDNA deletions. Although life expectancy was not shortened in our OPA1 mice, our observations also point to possible subclinical alteration in metabolism because Opa1 mice showed reduced body fat compared with controls.
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Accelerod test To determine fore and hind limb motor coordination and balance, a previously described accelerod test (Nguyen et al., 2006) was adapted for our mice. Briefly, this apparatus consisted of a base platform and a rotating rod with a non-skid surface. When operated in the acceleration modus, the rotor would accelerate from 4 to 40 rpm in a period of 5 min. Before testing, mice were trained four times a day for 2 min on three consecutive days. By this time, a steady baseline level of performance was attained. Opa1 mice (n = 13; 7 ♂ + 6 ♀) and controls (n = 6; 3 ♂ + 3 ♀) were tested at an age of 22 months starting with 4 rpm and subsequently accelerating after 30 s gradually up to 40 rpm in 5 min. The time spent on the rod before falling off was recorded and four independent experiments were averaged. To reveal the substantial variation of performance each result of the 4 single experiments has been included in the box-plots. mtDNA deletions The integrity of the mtDNA has been assessed as described previously (Tyynismaa et al., 2005) in DNA extracted (High Pure PCR Template Preparation Kit, Roche Diagnostics, Mannheim, Germany) from homogenate muscle tissue of 22-month-old mice. Briefly, long distance PCR (TaKaRa LA Taq, TaKaRa, Shiga, Japan) applying oligos: mito_2473-2505_for: 5′-GGT TCG TTT GTT CAA CGA TTA AAG TCC TAC GTG-3′ and mito_1953-1924_rev: 5′-GAG GTG ATG TTT TTG GTA AAC AGG CGG GGT-3′ was used to amplify a 16 kb fragment of mouse mtDNA in a 2 step cycling reaction (98°C for 10 s and 68°C for 12 min; 35 cycles). Histochemistry Observed differences between Opa1 mice and controls were considered significant with a p value b 0.05 and highly significant with a p value b 0.005 in an unpaired t-test. If not stated differently, all numbers are mean values ± standard deviation (SD). Kaplan–Meier plots were calculated for each group using SigmaPlot 2001 (SPSS, Chicago, Illinois, USA). Box plots show median, 25th and 75th percentiles as boxes, 10th and 90th percentiles as whiskers and outliners as dots. Statistics
Experimental methods Animals Mice were kept in a 12 h light (10 lx)/12 h dark cycle with food and water available ad libitum in full-barrier facilities free of specific pathogens. Mouse breeding and all experimental procedures were done according to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and approved by legal authorities. The Opa1enu/+ strain (abbreviated Opa1 mouse) has been initially described by our group elsewhere (Alavi et al., 2007). All examined mice were at least 22 months old.
The gastrocnemius muscle was harvested from the leg area of the following 22-month-old mice: Opa1+/+ (n = 5), non-affected Opa1+/- (n = 4) i.e. normal performance on the accelerod test, and affected Opa1+/- (n = 3), and immediately frozen in a melting isopentane bath (-150°C). Serial 20 μm muscle sections were cut and mounted onto glass slides using a Microm™ HM560 cryostat (Thermo Fisher, Germany), and these were then stained using established protocols for cytochrome c oxidase (COX), succinate dehydrogenase (SDH) and dual COX-SDH (Johnson and Barron, 1996, Taylor et al., 2004). Myofibrillar ATPase staining was also performed at a preincubation pH of 4.3 to further define the pattern of oxidative Type I (Dark) and glycolytic Type II (Light grey) fibers in these muscle sections (Johnson and Barron, 1996).
Primary phenotyping Results Thirty-nine aged Opa1 mice (24 ♂ + 15 ♀) and 15 age-matched controls (9 ♂ + 6 ♀) were first examined according to the modified SHIRPA protocol (European Mouse Phenotyping Resource of Standardised Screens; EMPReSS; http://empress.har.mrc.ac.uk/), which assesses mouse behavior in a viewing jar and above an arena. This screening was performed by two independent observers on 2 days. Furthermore, body weight and 24 h food intake were recorded for 32 Opa1 mice (20 ♂ + 12 ♀) and 10 (6 ♂ + 4 ♀) controls.
Primary SHIRPA screen We used the primary SHIRPA screen to assess the muscle and motor neuron function of a group of 39 22-month-old Opa1 mice and compared these results with those of 15 wild-type (WT) littermates (control). The Opa1 group included 15 females and 24 males (ratio ♀/♂ = 0.625) and hence was comparable to the control
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group with 6 females and 9 males (ratio ♀/♂ = 0.667). There were no significant differences for the following parameters recorded in a viewing jar: body position, palpebral closure, coat appearance, whiskers, lacrimation, defecation and skin color. One parameter differed significantly: 33% of the Opa1 mice showed an obvious resting tremor of the upper limbs versus none in the control group. With 2 ♀ vs. 11 ♂ there was also an imbalance toward males being more prone to the development of a tremor. The parameters of positional passivity, evidence of biting and vocalization that have been recorded above the arena, did not differ significantly, but 52% of the Opa1 mice showed an abnormal clasping of the hind limbs when suspended by their tail. There was no sex difference in terms of clasping, and none of the controls exhibited this behavior. Body weight and life span We found a highly significant difference in body weight. Body weights were determined for Opa1 mice with an average age of 635 days (SD = 30 days) and compared with controls with a mean age of 640 days (SD = 22 days; Fig. 1A). Under regular animal housing conditions, all animals in the control group were morbidly obese (mean: 48.9 g; SD = 7.2 g), while the Opa1 mice showed no signs of obesity (34.9 g; SD = 4.1 g; Figs. 1B and 2A). This was highly significant (p value = 1.9⁎10-9). Sex had only little effect on the body weight and differed within 1.6 g for both groups (not shown). There was no significant difference in food intake (Fig. 1C). A 24 h study revealed an average 24 h dry food intake of 4.7 g (SD = 0.5 g) for the controls and 4.4 g (SD = 0.9 g) for Opa1 mice (p value = 0.3597). Post mortem examinations revealed that the Opa1 animals had substantially less body fat compared to controls (Fig. 2A). Life expectancy was not reduced in the Opa1 group (mean life time: 765 days; SE = 25 days) when compared to the control group (758 days; SE = 19 days; Kaplan–Meier log rank test: p value = 0.42;
Fig. 1D). It did also not vary from data of a previous thorough study on the life expectancy of 746 C57Bl6 mice (Rowlatt et al., 1976). Accelerod test Motor coordination and balance capabilities of mice were measured using an accelerating Rotarod treadmill (accelerod) in an age-matched (mean age = 661 days; SD = 20 days) group of Opa1 animals and controls (mean age = 667 days; SD = 25). All control animals performed well in this experiment and acquired the task within the given time of training. In the Opa1 group some mice showed a clear disability to manage the task, while the remaining Opa1 animals' performance was indistinguishable from the control group. This is also reflected in the mean running time of the individual animals (Fig. 2B). While all controls taken together had a mean running time (mrt) of 88.7 s (SD = 26.4 s), Opa1 mice (mrt = 70.2 s; SD = 28.4 s) performed significantly worse (p value = 0.0352) than the controls (Fig. 2C; Control and Opa1 oa). If one would like to speculate, one could combine all Opa1 mice that had difficulties to manage the task (Opa1 aff.; mrt = 54.6 s; SD = 19.6 s) and compare it with the remaining Opa1 animals (Opa1 not aff.; mrt = 88.4 s; SD = 26.3 s; p value = 0.0001) or controls (p value = 0.0002; Fig. 2C). Noteworthy 3 out of 7 Opa1 animals which performed significantly worse in the accelerod experiments presented with tremor, whereas none of the well performing Opa1 animals had a tremor. The abnormal clutching reflex could be observed in 52% of the Opa1 animals independently of their performance in the accelerod test. Muscle morphology, COX activity and mtDNA integrity The Opa1 mouse has been considered as a model for nonsyndromic ADOA (Alavi et al., 2007), while certain forms of syndromic ADOA have been associated with mtDNA deletions (Amati-Bonneau et
Fig. 1. Opa1 mice show significantly less obesity than controls. Life expectancy is not altered for Opa1 mice. (A) Mean age of the mice ± SD at examination for weight and food intake. (B) Box plot of body weight, dashed line indicates the mean weight. (C) Mean food intake in 24 h of the mice ± SD. (D) Kaplan–Meier plot of the life expectancy for Opa1 and control mice (relative risk = 1.37; standard error 0.531; p value = 0.42).
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Fig. 2. Opa1 mice performed significantly worse in accelerod experiments. (A) Control mouse (left) and Opa1 mouse (right) on a treadmill. (B) Mean running time of 4 independent experiments for each single animal (error bars = SD). Last lane gives the mean running time of all animals of the according group. Opa1 mice performed significantly worse than controls. (C) Box plot of the accelerod performance for controls and Opa1 mice (Opa1 oa) depicting the single results of each run. Though speculative, if one combines all Opa1 mice that performed similar or better than the average (Opa1 not aff.; mouse 1 to 6 in B) and Opa1 mice that performed worse than the average (Opa1 aff.; mouse 7 to 13 in B), the Opa1 not aff. group performed similar to controls while the Opa1 aff. group performed highly significantly worse.
al., 2008; Hudson et al., 2008; Zeviani, 2008). Since our initial findings suggested subtle neuromuscular abnormalities, we analyzed the integrity of mtDNA in homogenate muscle samples derived from 22-month-old Opa1 mice and age-matched controls. We found minor mtDNA deletion bands in the majority of the analyzed muscle samples, from both Opa1 and control animals (Fig. 3A). However, all samples revealed a substantial content of intact mtDNA, which was not the case for homogenate muscle mtDNA derived from 18-monthold “deletor” mice, a model for progressive external ophthalmoplegia, which is caused by mutations in the mtDNA helicase twinkle that accumulates mtDNA deletions over time (Tyynismaa et al., 2005) (Fig. 3A, line 15). Histochemistry revealed normal muscle fiber morphology and no ragged red fibers (RRFs) or cytochrome c oxidase (COX) deficient fibers. Myofibrillar ATPase staining showed a preponderance of Type II fibers (90%–95%) with no observable pathological grouping of Type I fibers in mice of all 3 groups (Figs. 3B and C). Discussion The OPA1 gene is expressed ubiquitously (Alexander et al., 2000; Bette et al., 2005) and has been associated with fundamental processes such as mitochondrial fusion, apoptotic cristae remodeling and cytochrome c release (Cipolat et al., 2004, 2006; Frezza et al., 2006; Olichon et al., 2007). OPA1 homologs are known in yeast and all higher eukaryotes (Hales and Fuller, 1997). The importance of OPA1 is furthermore emphasized by the fact that mice with homozygous mutations in Opa1 are not viable and die during embryogenesis (Alavi
et al., 2007). In contrast the vast majority of heterozygous OPA1 mutations are associated with non-syndromic ADOA in humans (Ferre et al., 2005), that affect only the survival of RGCs. This led to the formulation of a fundamental question: What makes retinal ganglion cells unique that they are the only cells which are affected by OPA1 mutations? (Yu Wai Man et al., 2005). It has been hypothesized that the retina as highly oxygen consuming tissue and the optic nerve with its initial non-myelinated part might be especially prone to mitochondrial disturbances which would explain a mainly restricted ocular phenotype. However, can we exclude that OPA1 mutations do not affect other cell types or organs at subclinical levels? In other words: Why should heterozygous mutations in such a fundamental important gene like OPA1 impair solely the function of a single cell type? To address this question, we re-examined aged 22-month-old Opa1 mice (Alavi et al., 2007) for subtle phenotypic abnormalities. Using the primary SHIRPA screen, we found that about half of the Opa1 mice showed an abnormal clutching of their hind limbs. This indicates defects in brain, spinal cord or dorsal root ganglia (Klein et al., 1994; Mangiarini et al., 1996). One third of the Opa1 mice presented with a resting tremor of their upper limbs, suggesting defects of the brain stem or the cerebellum (Friedman et al., 2007). The Opa1 mice performed significantly worse in the accelerod experiment compared to controls, which points to neuromuscular coordination difficulties of the Opa1 mice (Nguyen et al., 2006). In summary, our experiments suggest that the subtle neuromuscular abnormalities observed in the mutant mice manifest relatively late in age and these are likely secondary to involvement of the peripheral
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Fig. 3. (A) Long distance PCR of muscle DNA from Opa1 mice and controls both demonstrated minor mtDNA deletions bands with the presence of a strong undeleted band representing the 16 kb amplicon. (B) ATPase staining with a pre-incubation pH of 4.3 showing a greater proportion of Type II fibers (light gray) compared to Type I fibers (dark), which is normal for mouse gastrocnemius muscle. There was no pathological grouping of Type I fibers. (C) Absence of COX-deficient fibers in muscle sections following dual COX–SDH histochemistry. The darker staining fibers are Type I due to their higher mitochondrial oxidative capacity compared to Type II fibers. These are representative slides from an affected 22-month-old Opa1 mouse but a similar staining pattern was observed in all mice studied. Bar = 200 μm.
and/or central nervous system. However we were not able to correlate the loss of retinal ganglion cells to the performance in the accelerod experiment, in other words a mouse with apparently advanced RGC loss performed very well in the accelerod, while a mouse with minor RGC loss performed worse. See Heiduschka et al. 2009 for a full description of the ocular phenotype. Recent publications demonstrate the accumulation of mtDNA deletions in patients with syndromic forms of OPA1-associated optic atrophy that suffer from severe additional neurological symptoms like sensorineural deafness, ataxia, axonal sensory-motor polyneuropa-
thy, chronic progressive external ophthalmoplegia and mitochondrial myopathy (Amati-Bonneau et al., 2008; Ferraris et al., 2008; Hudson et al., 2008). However histological analyses of muscle sections from Opa1 mice excluded a primary myopathic process and we did not find any evidence for the presence of significant amounts of deleted mtDNA molecules in our muscle samples. The detected minor deletion bands were comparable between Opa1 mice and controls and might originate from normal ageing processes rather than represent a pathologic finding (Taylor et al., 2004). This finding would suggest that our Opa1 mouse is a model for non-syndromic ADOA rather than one for syndromic ADOA. In this context it is of particular importance that the Opa1 mouse does not suffer from any hearing impairment (Alavi et al., 2007). There was also no evidence of COX-deficient fibers and with a minimum sampling of 1000 muscle fibers for each mouse, if COX-deficient fibers were missed, these are likely be present at only low levels (b0.5%) and therefore of no pathological significance. Type II fibers constitute N90% of all fibers in mouse gastrocnemius muscle (Hayasaki et al., 2001; Manttari and Jarvilehto, 2005) and based upon myofibrillar ATPase staining, we observed a similar distribution in our Opa1 mice, irrespective of mutational or neuromuscular status. Importantly, angulated fibers were absent and there was no evidence of pathological grouping of oxidative Type I fibers precluding a denervation process affecting the peripheral nerves. The normal fiber architecture and absence of inflammatory cells on histology also excluded the possibility of an underlying inflammatory or degenerative myopathic process. Therefore we would like to postulate that the pathology is central in origin because we can exclude a myopathy and a peripheral neuropathy, and although we have no data at this stage we would like to speculate about a possible cerebellar origin. The difference in body weight of Opa1 mice compared to the obese controls might indicate an altered metabolism in aged mutant mice, supported by the reduced body fat content in the mutant mice. Although speculative, altered insulin secretion might lead to less accumulation of body fat over time, given the recent findings that the Opa1 protein is involved in mitochondrial maintenance in pancreatic beta cells (Twig et al., 2008). Future experiments are required to investigate metabolic function in Opa1 mice in more details. Only about half of aged animals showed these phenotypic abnormalities. Moreover, these additional phenotypic features do not appear to be linked with the actual severity of RGC loss and hence level of visual impairment. Since the Opa1 mouse strain is not congenic inbred this phenotypic variability points to the presence of genetic modifiers which is in line with the observed strong ocular phenotypic variability in humans (Fuhrmann et al., 2009). Outcross to different genetic backgrounds might help to identify new modifier genes in the future. Taken together, we have shown that RGCs are not the only cell types which are affected by Opa1 mutations in an Opa1 mouse model for ADOA. Moreover, our findings indicate that ADOA with additional, though subclinical extra-ocular manifestations is not necessarily linked with the presence of multiple mtDNA deletions, highlighting the need to further elucidate the other roles played by the OPA1 protein in normal mitochondrial function. Therefore this study complements a recent report on the absence of mtDNA defects in atrophic optic nerves of a second Opa1 mouse model (Yu-Wai-Man et al., 2009). Recently, a subclinical neuromuscular phenotype in an ADOA family without any mtDNA deletions has been reported (Spinazzi et al., 2008), which is in line with our findings. Therefore, we consider it possible that mutations in OPA1 can cause additional subtle neuromuscular impairments even in apparently non-syndromic ADOA patients, because of the crucial role of the ubiquitously expressed OPA1 protein in mitochondrial biogenesis and maintenance, and its involvement in fundamental cellular functions. We therefore want to encourage clinicians to be more attentive to extraocular manifestations in patients with optic atrophy.
Brief Communication Conflict of interest statement The authors wish to declare no competing interest.
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