J Neurol DOI 10.1007/s00415-015-7696-5

ORIGINAL COMMUNICATION

Distributed abnormalities of brain white matter architecture in patients with dominant optic atrophy and OPA1 mutations Maria A. Rocca • Stefania Bianchi-Marzoli • Roberta Messina • Maria Lucia Cascavilla • Massimo Zeviani • Costanza Lamperti • Jacopo Milesi • Arturo Carta • Gabriella Cammarata Letizia Leocani • Eleonora Lamantea • Francesco Bandello • Giancarlo Comi • Andrea Falini • Massimo Filippi

•

Received: 15 December 2014 / Revised: 27 February 2015 / Accepted: 27 February 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract Using advanced MRI techniques, we investigated the presence and topographical distribution of brain grey matter (GM) and white matter (WM) alterations in dominant optic atrophy (DOA) patients with genetically proven OPA1 mutation as well as their correlation with clinical and neuro-ophthalmologic findings. Nineteen DOA patients underwent neurological, neuro-ophthalmologic and brainstem auditory evoked potentials (BAEP) evaluations. Voxel-wise methods were applied to assess regional GM and WM abnormalities in patients compared to 20 healthy controls. Visual acuity was reduced in 16 patients. Six DOA patients (4 with missense mutations) had an abnormal I peripheral component (auditory nerve) at BAEP. Compared to controls, DOA patients had significant atrophy of the optic nerves (p \ 0.0001). Voxel-based morphometry (VBM) analysis showed that, compared to

controls, DOA patients had significant WM atrophy of the chiasm and optic tracts; whereas no areas of GM atrophy were found. Tract-based spatial statistics (TBSS) analysis showed that compared to controls, DOA patients had significantly lower mean diffusivity, axial and radial diffusivity in the WM of the cerebellum, brainstem, thalamus, fronto-occipital-temporal lobes, including the cingulum, corpus callosum, corticospinal tract and optic radiation bilaterally. No abnormalities of fractional anisotropy were detected. No correlations were found between volumetric and diffusivity abnormalities quantified with MRI and clinical and neuro-ophthalmologic measures of disease severity. Consistently with pathological studies, tissue loss in DOA patients is limited to anterior optic pathways reflecting retinal ganglion cell degeneration. Distributed abnormalities of diffusivity indexes might reflect abnormal

M. A. Rocca
R. Messina
M. Filippi (&) Neuroimaging Research Unit, Institute of Experimental Neurology, Division of Neuroscience, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Via Olgettina, 60, 20132 Milan, Italy e-mail: [email protected]

M. Zeviani MRC Mitochondrial Biology Unit, Cambridge, UK

M. A. Rocca
R. Messina
L. Leocani
G. Comi
M. Filippi Department of Neurology, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy S. Bianchi-Marzoli
G. Cammarata Department of Ophthalmology, Neuro-ophthalmology Unit, IRCCS Istituto Auxologico Italiano, Milan, Italy

A. Carta Department of Biomedical, Biotechnological and Translational Sciences (S.Bi.Bi.T), Ophthalmology Unit, University of Parma, Parma, Italy L. Leocani Experimental Neurophysiology Unit, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy A. Falini Department of Neuroradiology, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy

M. L. Cascavilla
J. Milesi
F. Bandello Department of Ophthalmology, San Raffaele Scientific Institute, Vita-Salute San Raffaele University, Milan, Italy M. Zeviani
C. Lamperti
E. Lamantea Unit of Molecular Neurogenetics, Foundation ‘‘C. Besta’’ Neurological Institute-IRCCS, Milan, Italy

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intracellular mitochondrial morphology as well as alteration of protein levels due to OPA1 mutations. Keywords Dominant optic atrophy
Optic coherence tomography
Magnetic resonance imaging
Regional atrophy
Diffusion tensor MRI
White matter
Mitochondrial mutation

Introduction Autosomal dominant optic atrophy (DOA) is the most prevalent hereditary optic neuropathy typically presenting with a slowly progressive, painless, bilateral visual loss in the first two decades of life, with centrocecal scotoma, impaired colour vision and bilateral temporal or diffuse atrophy of the optic nerve [1]. The clinical expression of the disease is extremely variable, with incomplete penetrance in some families. Post-mortem studies have shown a primary degeneration of retinal ganglion cells, followed by ascending atrophy of the optic nerve [2, 3]. The main DOA-causing gene is the nuclear OPA1 gene, which encodes a mitochondrial GTPase located in the intermembrane space involved in mitochondrial fusion, cristae organisation and control of apoptosis [4, 5]. The OPA1 protein is also associated with mitochondrial DNA maintenance [6, 7]. The mutation spectrum includes missense, nonsense, small deletion/insertion, splicing mutations and genomic rearrangements distributed throughout the gene on chromosome 3. The majority of OPA1 mutations generates a truncated protein leading to haploinsufficiency as pathogenic mechanism [8]. Similarly to other hereditary optic neuropathies (e.g., Leber hereditary optic neuropathy-LHON), extra-ocular neurological complications have been described in a variable percentage of mutational carriers (up to 20 % of the cases) [6, 9], including bilateral sensorineural deafness beginning in late childhood, ataxia, myopathy, peripheral neuropathy, external ophthalmoplegia, spastic paraparesis (mimicking hereditary spastic paraplegia) and a multiple sclerosis-like illness. These multi-systemic phenotypes have been associated to the presence of cytochrome c oxidase-deficient fibres and multiple mitochondrial DNA deletions in skeletal muscle [6, 9]. In patients with primary or secondary optic neuropathies of different aetiologies, magnetic resonance imaging (MRI) techniques are contributing to define the presence and distribution of brain damage as well as the possible underlying pathological substrates. At present, it is still unclear to what extent the central nervous system (CNS) is affected in patients with DOA and, most importantly, it has not been evaluated yet whether such an involvement is restricted to the optic nerve and visual pathways, as it has been described in optic

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neuritis [10, 11], chronic glaucoma [12] and retinal degeneration [13], or conversely affects in a more distributed pattern the brain white matter (WM) and grey matter (GM), as it has been shown for other hereditary ocular pathologies with mitochondrial dysfunctions, such as LHON [14, 15]. To answer the previous unsolved question, in this study we applied voxel-wise techniques to define the presence and topography of WM and GM involvement in patients with DOA. Specifically, irreversible tissue loss was explored by means of voxel-based morphometry (VBM), while abnormalities of architectural organisation of the WM were investigated with tract-based spatial statistics (TBSS). To provide some clues about the nature of the detected changes (if present), we also assessed their correlation with the duration of the disease, the severity of visual impairment and the extent of retinal damage, quantified using visual acuity and optic coherence tomography (OCT), respectively.

Materials and methods Subjects We studied 19 patients with DOA (10 women, 9 men; mean age = 43.2 years, range = 22–64 years; mean disease duration = 26 years, range = 2.4–55 years) from 11 independent families recruited consecutively from the NeuroOphthalmology Clinics at the San Raffaele Scientific Institute, Milan; the Neuro-Ophthalmology Unit of the Ophthalmology Department, IRCCS Istituto Auxologico Italiano, Milan, and the Hospital of Parma, Italy. Inclusion criteria were: (a) presence of one of the pathogenic heterozygous mutations in the OPA1 gene associated with DOA, (b) no history of concomitant neurological, psychiatric, major medical conditions, or substance abuse, and (c) no other ophthalmic diseases (apart from sequelae secondary to optic nerve involvement). At the time of MRI acquisition, all patients were evaluated by an expert neurologist and, except from the visual deficits, they did not have any neurological symptoms or signs neither complaint of cognitive deficits. Twenty age- and sex-matched healthy subjects (10 women, 10 men; mean age = 42.7 years, range = 25–59 years) with no history of neurological and ophthalmological disorders and a normal neurological and ophthalmological examination served as controls. Approval from the local ethics committee and written informed consent from all subjects were obtained before study initiation. Neuro-ophthalmologic assessment Before MRI acquisition, each patient underwent a complete neuro-ophthalmologic examination. Best-corrected visual

J Neurol

acuity (BCVA) was assessed with logMAR notation performed with high-intensity red-free light. Visual field assessment was performed with standardised automated perimetry (SAP) and Mean Deviation was quantified (Humphrey Zeiss, 30-2 SITA standard program). For each eye, average peripapillary retinal nerve fibre layer (PRNFL) and ganglion cell complex (GCC) thickness measurements were obtained using a commercially available optical coherence tomography (OCT) (Optovue, RTVue 100). Pupil dilatation was induced in all patients, and an internal fixation was used whenever possible. The OCT software uses an automated computerised algorithm to rank the PRNFL and GCC thickness measurements from each patient’s eye against a normal percentile distribution derived from a database of age-matched control subjects. For this reason, OCT measurements were not obtained from the healthy controls of this study. Neurophysiologic assessment Brainstem auditory evoked potentials (BAEPs) were recorded with frontopolar ground electrode in a quiet room from all DOA patients. The BAEPs to clicks at 80 dB normal hearing level were recorded at the Cz electrode referred to the ipsilateral and contralateral ear. The latency of the main peaks I and V were measured. If the I wave was delayed or absent with 80 dBnHL, the stimulation intensity was increased in steps of 10 dB up to 100 dBnHL. For each side, absolute and interpeak latencies of the I (acoustic nerve) and III, V (brainstem) waves were measured and compared with normative data obtained at the same laboratory; an interside asymmetry exceeding 0.3 ms was considered abnormal, as well as a V/I amplitude ratio lower than 0.5. MRI acquisition Using a 3.0 Tesla Intera scanner (Philips Medical Systems, Best, The Netherlands), the following sequences of the brain were obtained from all subjects: (1) axial T2weighted turbo-spin echo (repetition time (TR)/echo time (TE) = 3000/120 ms, flip angle (FA) = 908, matrix size = 512 9 512, field of view (FOV) = 230 mm2, 28.4 mm thick, contiguous slices); (2) axial fluid-attenuated inversion recovery (FLAIR) (TR/TE = 11000/ 120 ms, inversion time = 2800 ms, FA = 90°, matrix size = 256 9 256, FOV = 230 mm2, 28.4 mm thick, contiguous slices); (3) axial 3D T1-weighted fast field echo (FFE) (TR/TE = 25/4.6 ms; flip angle = 30°; matrix size = 256 9 256; FOV = 230 9 230 mm2; 220 contiguous slices with voxel size = 0.89 9 0.89 9 0.8 mm); and (4) pulsed-gradient spin-echo, echo-planar (TE/ TR = 58/8775.5 ms; flip angle = 90°; matrix size =

96 9 96; FOV = 240 9 240 mm2; 55 contiguous, 2.3 mm thick axial slices) with SENSE (acceleration factor = 2) and diffusion gradients applied in 35 non-collinear directions. Two optimised b factors were used for acquiring diffusion-weighted images (b1 = 0, b2 = 900 s/mm2). MRI analysis MRI analysis was performed by a single neurologist, who was unaware of subjects’ identity. T2-weighted scans were analysed for the presence of lesions and FLAIR scans were always used to increase confidence in their identification. Lesion volumes (LV) were measured using a local thresholding segmentation technique (Jim 5.0, Xinapse System, Leicester, UK). Using the same technique, the volume of the intraorbital part of the optic nerves was also quantified. To this aim, regions of interest (ROIs) including the optic nerves were identified on coronal slices from high-resolution 3D T1-weighted sequences of the brain, starting from the first coronal slice behind the retina to the optic canal, as previously described [16]. The mean intraobserver variability for optic nerve volume quantification using this methods was 1.5 % (range = 0.06–4 %) [16]. On 3D T1-weighted images, normalised brain volumes (NBV) were calculated using the cross-sectional version of the Structural Imaging Evaluation of Normalised Atrophy (SIENAx) software [17]. Distribution of regional atrophy of the WM and GM VBM analysis was performed using SPM8 (www.fil.ion. ucl.ac.uk/spm), as previously described [18, 19]. First, FFE images were segmented into GM, WM and CSF. Then, GM and WM segmented images of all subjects, in the closest possible rigid-body alignment with each other, were used to produce GM and WM templates and to drive the deformation to the templates. To produce the templates, at each iteration, the deformations, calculated using the Diffeomorphic Anatomical Registration using Exponentiated Lie algebra (DARTEL) registration method [20], were applied to GM and WM, with an increasingly good alignment of subject morphology. Spatially normalised images were then modulated to ensure that the overall amount of each tissue class was not altered by the spatial normalisation procedure. To better align the final template with the Montreal Neurologic Institute (MNI) space, an affine registration between the customised GM template and the SPM GM template (in the MNI space) was also calculated and added to the header of each image as a new orientation, to have all images in a standard space. The same transformation was applied to the WM customised template. The images were then smoothed with an 8 mm FWHM Gaussian kernel.

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Distribution of regional DT MRI damage to WM (TBSS) Diffusion-weighted images were first corrected for distortions caused by eddy currents. Then, using the FMRIB’s Diffusion Toolbox (FDT tool, FSL 4.1, http://www.fmrib. ox.ac.uk), the diffusion tensor (DT) was estimated in each voxel by linear regression [21] and fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity (RD) maps derived. TBSS analysis was used to perform a voxel-wise analysis of the whole brain WM DT MRI measures (http://www.fmrib.ox.ac.uk/fsl/ tbss/index.html). In detail, individual FA images were nonlinearly registered to the FMRIB58_FA atlas provided within FSL, and averaged to obtain a customised atlas. To optimise inter-subject comparability, this step was performed again by using the population-derived atlas as a target. The resulting FA atlas was then thinned to create a WM tract ‘‘skeleton’’, which was thresholded at a FA [ 0.2 to include WM voxels only. Individual subject normalised FA maps were warped onto the FA skeleton before statistical analysis, by searching perpendicularly from the skeleton for maximum FA values [22]. The individual registration and projection vectors obtained during this process were also applied to MD, AD and RD maps. Statistical analysis Normal distribution assumption was checked for continuous variables by using Kolmogorov-Smirov and Shapiro–Wilk tests, as well as with graphical inspection of Q–Q plots. Between-group comparisons were performed using parametric and non-parametric tests as appropriate. Categorical variables were compared between groups using the Fisher exact test (SPSS software, version 21.0). Between-group comparisons of smoothed GM and WM maps were assessed using analyses of covariance, including age and the normalisation factor derived from SIENAx (which can be considered as a measure of head size) as covariates. Results were assessed at a threshold of p \ 0.05, family-wise error (FWE) corrected for multiple comparisons. Voxel-wise differences of FA, MD, AD and RD values between DOA patients and controls were tested using a permutation-based inference for non-parametric statistical thresholding (‘‘randomise’’ program within FSL) [23] and two-sample t tests, adjusting for age. The number of permutations was set to 5.000. The resulting statistical maps were thresholded at p \ 0.05, with correction for multiple comparisons (FWE corrected) at a cluster level using the threshold-free cluster enhancement [24]. The WM tracts were identified using WM atlases provided within FSL [25–27].

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In DOA patients, correlations between optic nerve volumes, regional GM/WM volumes and DT MRI abnormalities vs. disease duration (defined as the time elapsed from the onset of visual symptoms or the detection of optic disc pallor at neuro-ophthalmologic evaluation), neuroophthalmologic (BCVA, Mean Deviation, average PRNFL and GCC thickness) and neurophysiologic (latency of the main peaks I and V) findings were assessed using SPSS, SPM8 and FSL, as appropriate. The correlation between optic nerve volumes vs. regional GM/WM volumes and DT MRI abnormalities was also evaluated.

Results Clinical and neuro-ophthalmologic assessment The main demographic, clinical, genetic, and neuro-ophthalmologic characteristics of DOA patients are summarised in Table 1. Seven (37 %) DOA patients had missense mutations. At the time of MRI acquisition, 16 patients had a bilateral visual impairment and 3 patients had no visual acuity loss. LogMAR BCVA was decreased in 32 (84 %) affected eyes, mean LogMAR BCVA was = 0.59, range from 2 to 0 (p = 0.45 for comparison between right and left eye, Wilcoxon signed-rank test). Average Mean Deviation was = -6.5 dB, range from -30.8 to -0.98 dB (p = 0.29 for comparison between right and left eye, Wilcoxon signed-rank test). Average PRNFL thickness was reduced in 35 (92 %) affected eyes and GCC thickness in 31 (82 %) affected eyes: mean PRNFL thickness was 69.84 lm, range from 53 to 88 lm (p = 0.65 for comparison between right and left eye, paired t test); mean average GCC thickness was 65.88 lm, range from 43 to 88.5 lm (p = 0.65 for comparison between right and left eye, paired t test). Mean Deviation abnormalities were significantly more pronounced in patients with missense mutations vs those with mutations causing haploinsufficiency (p = 0.04); the remaining clinical and neuro-ophthalmologic measures did not differ between the two groups of subjects. Neurophysiological assessment None of the patients complained of auditory symptoms at the time of testing. BAEPs were normal in 13 DOA patients (68 %; 3 with missense mutations). Six DOA patients (32 %, of whom 3 siblings, 4 with missense mutations) had an abnormal I peripheral component (auditory nerve): four of them had mild/moderate abnormalities (bilateral in one patient only) consisting in a delay or absence of the peripheral I component at 80 dBnHL, with normalisation at higher intensities (90–100 dBnHL).

M

F

F

F M

M F

M

F

F

M

M

M

F

M

2a

3a

4a

5a 6a

7a 8

9

10

11

12a

13

14

15

16

F

M

1

17

Sex

Subject

49

64

42

46

28

22

39

45

48

27 44

32 63

44

53

56

34

Age

34

39

12

25

20

10

28

6

12

4 25

6 8

11

14

10

22

Age of onset

No

Yes

Yes

No

No

No

No

No

No

Yes No

Yes Yes

Yes

Yes

Yes

No

Family history

p.(Arg857*) p.? p.?

c.2569C [ T Exon_25 c.2708-1G [ T Intron_26 c.2708-1G [ T Intron_26 c.870 ? 5G [ A Intron_8

Splice defect

p.(Glu595*)

p.(Val933Ile)

p.?

p.(Val903Glyfs*3)

Splice defect

p.(Val910Asp) p.(Val903Glyfs*3)

p.(Asp470Gly) p.(Val910Asp)

p.(Asp470Gly)

p.(Asp470Gly)

p.(Asp470Gly)

p.(Val942Glufs*25)

Protein change (isoform 1) NP_056375.2

c.1782_1783insT Exon_19

c.2797G [ A Exon_27

c.33-?_678 ? ?dup Exon_2_6

c.2708_2711delTTAG Exon_27

c.1770 ? 1delG Intron_18

c.2729T [ A Exon 27 c.2708_2711delTTAG Exon_27

c.1409A [ G Exon_14 c.2729T [ A Exon 27

c.1409A [ G Exon_14

c.1409A [ G Exon_14

c.1409A [ G Exon_14

c.2823_2826delTTAG Exon_28

OPA1 mutation (NM_015560.2)

N/N

M/M

N/N

N/N

N/N

M/S

N/N

N/N

N/N

N/N N/M

N/N N/N

N/M

N/M

S/S

N/N

-4.2

-9.5

-1.5

-3.1

-1.8

-33.1

-6.8

-2.3

-3.3

-10.4 -3.3

-2.3 -4.5

-30.5

-2.2

-5.4

-1.3

Right eye

Better side/worse side

-3.3

-10.5

-1.8

-3.3

-2.1

-13.3

-7.4

-2.4

-4.9

-9.7 -3.7

-3.5 -3.5

-31.2

-1.9

-5.9

-2.6

Left eye

Average mean deviation (dB)

BAEP

0.5

1.3

0

0.7

0.4

0.1

0.7

0.4

0.7

1 0.3

0.7 0.7

1.3

0.22

1.3

0.4

Right eye

0.7

1.52

0

0.4

0.4

0.22

0.5

0.4

1

1.3 0.5

0.5 0.22

1.3

0.22

1.6

0.4

Left eye

LogMAR BCVA

75

63

79

65

64

98

62

70

75

68 74

69 64

60

70

52

81

Right eye

70

60

79

71

63

59

64

66

81

70 81

81 68

54

69

54

76

Left eye

Average PRNFL thickness (lm)

Table 1 Clinical, demographic, neurophysiological and conventional magnetic resonance imaging data of the patients enrolled in the study

85

62

73

77

60

62

56

63

82

71 82

47 67

53

55

44

73

Right eye

83

59

66

71

63

57

55

58

95

71 95

48 72

52

56

43

75

Left eye

Average GCC thickness (lm)

Non-specific brain WM lesions and Non-specific brain WM lesions

Non-specific brain WM lesions

Normal

Normal

Non-specific brain WM lesions

Non-specific brain WM lesions

Normal

Diffuse WM hyperintensities in the ORs

Normal Non-specific brain WM lesions

Normal Non-specific brain WM lesions and focal lesion in the ORs

Non-specific brain WM lesions

Normal

Non-specific brain WM lesions

Normal

Brain MRI findings

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The remaining two patients had severe I abnormality, being absent even at higher intensity (100 dBnHL). None of the patients had abnormal central conduction parameters (absolute and interpeak latencies of central components, V/I amplitude ratio). Overall, the observed BAEPs abnormalities were compatible with hearing loss of peripheral origin.

Patients with missense mutations

MRI assessment

a

N normal, M mild or moderate (peripheral wave only delayed or not defined at 80 dBnHL, normal at higher intensities), S severe abnormalities (wave I absent up to 90/100 dBnHL), BAEP brainstem auditory evoked potentials, BCVA best-corrected visual acuity, PRNFL peripapillary retinal nerve fibre layer, GCC ganglion cell complex, MRI magnetic resonance imaging, WM white matter, OR optic radiation

Mutational data are described using the nomenclature of the Human Genome Variation Society (http://www.hgvs.org/mutnomen). Nucleotide numbering reflects complementary DNA numbering with ?1 corresponding to the A of the ATG translation initiation codon in the reference sequence (human OPA1, RefSeq: NM_015560.2). The initiation codon is codon 1

Normal

Normal

58

77

52

80

59 58

89 0

0 0

0 -1.2

-4.3 -3.8

-0.8 N/N

N/N p.(Arg52*)

p.(Gln785Serfs15*)

c.154C [ T Exon_2

c.2353delC Exon_23 Yes

No 3

39 41

F

F

18

19

41

87

Left eye Right eye Left eye Right eye Left eye Right eye Right eye Better side/worse side

Protein change (isoform 1) NP_056375.2 OPA1 mutation (NM_015560.2) Family history Age of onset Age Sex Subject

Table 1 continued

Left eye

Average mean deviation (dB) BAEP

LogMAR BCVA

Average PRNFL thickness (lm)

Average GCC thickness (lm)

Brain MRI findings

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Non-specific brain focal T2-hyperintense lesions located in subcortical and/or periventricular regions were detected in 10 (53 %) DOA patients and six (30 %) healthy controls. One DOA patient had focal T2 lesions close to the optic radiation (OR) and another one showed distributed WM hyperintensities along the OR (Fig. 1). Mean brain T2 LV was 0.7 ml (SD = 1.81 ml) in DOA patients and 0.03 ml (SD = 0.06 ml) in healthy controls (p = 0.04). Mean NBV was 1540 ml (SD = 48 ml) in DOA patients and 1551 ml (SD = 96 ml) in healthy controls (p = 0.64). Since right and left optic nerve volume did not differ significantly nor in DOA patients (right optic nerve = 0.13 ml and left optic nerve = 0.12 ml, p = 0.3) neither in controls (right optic nerve = 0.19 ml and left optic nerve = 0.20 ml, p = 0.8), their mean values entered the statistical analyses. Compared to controls, DOA patients had significant atrophy of the optic nerves (mean optic nerve volume: 0.20 ml, SD = 0.05 in healthy controls and 0.13 ml, SD = 0.5 in DOA patients, p \ 0.0001) (Fig. 1). Optic nerve atrophy was significantly more pronounced in patients with missense mutations vs those with other mutations causing haploinsufficiency (p = 0.01). VBM analysis showed that, compared to controls, DOA patients had significant WM atrophy of the chiasm and optic tracts (MNI coordinates: -6, 7, -19, cluster extent = 117, p \ 0.05 FWE corrected) (Fig. 2). No areas of GM atrophy were found in DOA patients compared to healthy controls, even when using an uncorrected statistical threshold. Similar results (with a different level of significance) were found when comparing patients with missense mutations and those with other mutations vs healthy controls. No differences were found between the two groups of patients (data not shown). TBSS analysis showed that, compared to controls, DOA patients had a distributed pattern of decreased MD and RD of the WM (p \ 0.05 FWE corrected) involving the corpus callosum (CC), cerebellum, corticospinal tract (CST), cingulum, thalamic radiation, OR, acoustic radiation, superior (SLF) and inferior longitudinal fasciculus (ILF), inferior fronto-occipital fasciculus (IFOF) and brainstem, bilaterally. Decreased WM AD (p \ 0.05, FWE corrected) of the genu of the CC, right ILF, cerebellum, CST, thalamic radiation, OR, acoustic radiation and brainstem,

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bilaterally, was also found (Fig. 3). No WM FA abnormalities were detected. Compared to patients with mutations possibly associated to haploinsufficiency, those with missense mutations had significantly lower WM AD (p \ 0.05, FWE corrected) of the cerebellum and brainstem, as well as the left CST, thalamic radiation, OR and acoustic radiation. Analysis of correlations In DOA patients, no correlation was found between regional WM volumes and DT MRI abnormalities vs disease duration, optic nerve volumes, neuro-ophthalmologic and neurophysiologic findings, even when analysing patients in whom haploinsufficiency is supposed as a pathogenic mechanism and those with missense mutations separately.

Discussion Using advanced MRI techniques, in this study we mapped the regional distribution of damage to the CNS in a

Fig. 1 a An illustrative axial FLAIR magnetic resonance image (MRI) from a 63-year-old man affected by dominant optic atrophy (DOA), which shows focal lesions in the subcortical white matter (WM) and along the optic radiations (ORs) (arrows); b An illustrative axial FLAIR MRI from a 48-year-old man with DOA, which shows distributed T2 hyperintensities along the ORs (arrow); c An

relatively large group of DOA patients with OPA1 genetically confirmed mutation and the relation with disease duration and severity of visual impairment. The application of a similar approach to patients affected by other forms of hereditary or acquired optic neuropathies has allowed to demonstrate, in vivo, structural abnormalities along the visual pathways, extending from the optic nerve to the retrochiasmatic structures and visual cortex. To assess optic nerve involvement, we used OCT measurements of PRNFL and GCC thickness as well as MRI quantification of the volumes of both optic nerves. In line with the results of previous neuro-ophthalmologic studies and with the well-known preferential involvement of small fibres in the papillomacular bundles (an hallmark of mitochondrial optic neuropathies), the majority of our patients experienced bilateral visual impairment, with a reduction of both PRNFL and GCC [28, 29]. The few pathological studies that have been performed in DOA patients so far have shown ascending optic nerve atrophy, as a consequence of a primary degeneration of the ganglion cell layer in the retina [2, 3]. This is consistent with the severe reduction of optic nerve volume found in our

illustrative coronal 3D T1-weighted MRI from a 32-year-old healthy man, which shows a normal optic nerves volume (ONV); d An illustrative coronal 3D T1-weighted MRI from a 56-year-old man with DOA, which shows a bilateral optic nerve atrophy. Contours of the optic nerves used to assess ONV are shown in red

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Fig. 2 In the front, areas showing significant white matter atrophy (p \ 0.05, FWE corrected) in patients with dominant optic atrophy (DOA) compared to healthy controls, superimposed on the patients’ customised white matter (WM) template. Atrophy of the chiasm and

the optic tracts was found in patients with DOA. In the background, the patients’ customised WM template, including the chiasm and optic tracts is shown. L left, R right

and previous studies [30]. Previous studies have also demonstrated a reduced optic nerve size in DOA patients [31]. This, combined with the finding that age-dependent loss of RNLF thickness is preserved in those quadrants with higher residual amount of fibres whereas it is lost in fibre depleted quadrants, has led to the conclusion that fewer optic nerve axons are present from birth in these patients [28]. Although ocular manifestations were the only subjective complaint in our DOA cases, BAEP abnormalities were present in six subjects, four of whom carrying a missense mutation. Sensorineural hearing loss is the second most frequent manifestation of DOA associated with OPA1 mutation, having being described in a frequency between 6 and 20 % of the cases [9, 32]. Such an involvement is not unexpected, considering that OPA1 is also expressed in inner hair cells and spiral ganglion cells of the cochlea and vestibular organ [33]. Noteworthy, a decreased number of neurofibrils and myelin sheaths have been described in the intracranial part of both vestibulocochlear nerves in one of the available post-mortem studies of a patient with DOA [3]. At present, the exact prevalence of hearing defects in DOA patients is likely to be underestimated. This is because from the one hand different type of mutations associated with different defects of mitochondrial function and with a different penetrance are progressively being discovered. On the other hand, the clinical deficits might be congenital or slowly developing and become clinically meaningful and thus undergo genetic testing only when they are severe. It is important to mention that electrophysiological evidence of auditory dysfunction in patients with hereditary optic neuropathies seems not to be limited to OPA1 mutation carriers, since it has also been shown in

other mitochondrial diseases, such as LHON [34] and Friedreich’s ataxia [35]. The MRI analysis of the regional distribution of atrophy showed that DOA patients had a selective tissue loss of the anterior visual pathways, which did not spread to the ORs and visual cortex. Such a pattern of tissue loss along the visual pathways differs from those described in other forms of hereditary or acquired optic neuropathies (such as LHON, glaucoma and demyelinating optic neuritis) [10– 12], in which atrophy of the posterior optic pathways has been shown following damage to the retina and optic nerve, which is likely secondary to trans-synaptic degeneration phenomena. Among the factors which might contribute to explain differences of regional distribution of damage between DOA due to OPA1 mutations and LHON patients, we need to consider not only the diversity of molecular defects and genetic inheritance, but also of clinical onset and visual defect severity. Indeed, differently from LHON, which typically presents with rapid and severe loss of central vision, patients with DOA have slowly progressive bilateral loss of central vision, which is frequently mild [36]. Our volumetric findings are in line with the pathological study of Kjer and coworkers, who described tissue loss limited to the anterior optic pathways in a DOA patient, without involvement of the visual cortex [3]. Remarkably and differently from this pathology study [3], which found tissue loss and fibrillary gliosis of the lateral geniculate nucleus (LGN), our VBM analysis disclosed no GM loss in this structure, even when the statistical threshold for significance was lowered at a p \ 0.001, uncorrected. Several factors might contribute to explain this somewhat unexpected finding. First, the papillomacular bundle, which is

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Fig. 3 Areas of significantly reduced white matter (WM) mean (red), axial (green) and radial (yellow) diffusivity (p \ 0.05, FWE corrected) in patients with dominant optic atrophy (DOA) vs. healthy

controls, superimposed on a fractional anisotropy template in the Montreal Neurological Institute space. MD mean diffusivity, AD axial diffusivity, RD radial diffusivity, L left, R right

hit in DOA patients, represents only a part of the projections from the eyes to the LGN. In addition, the involvement of this bundle in DOA patients may be limited to some fibres, with a preferential affection of the superiortemporal sectors. This limited damage might be beyond the present resolution offered by MRI. An alternative explanation for the lack of LGN atrophy might be the coexistence in this structure of gliosis and lipid deposition, which may ‘‘hide’’ tissue loss. The most intriguing finding of our study is the in vivo demonstration of distributed abnormalities of diffusivity indexes in the WM skeleton, which are characterised by decreased MD, RD and AD not only along the visual pathways, but also in the majority of the remaining hemispheric and cerebellar WM tracts. Conceptually such a distributed involvement of the WM is not unexpected, considering the ubiquitous expression of OPA1 protein in the brain, including neurons of motor and frontal cortex, cerebellar cortex and astrocytes [37]. OPA1 is imported into mitochondria and contributes to structuring cristae, cristae function and the interaction between outer and inner mitochondrial membranes, being important for the

maintenance of the morphology of intracellular mitochondrial network. Down-regulation of OPA1 leads to fragmentation of mitochondria, mitochondrial dysfunction (e.g., destabilisation of the respiratory chain supercomplexes) [38], altered mitochondrial inner membrane morphology (from tubular to vesicular) [39], and increased propensity for apoptosis [39]. Dysfunctional mitochondria modify not only their architecture, but also their distribution and density within cells and their dendrites, thus possibly contributing to the DT MRI abnormalities we found. An additional mechanism that is likely at work in causing reduced MD, AD and RD might be related to OPA1 abnormalities per se, which from one hand lead to reduction of synaptic protein expression levels and alterations of dendritic arborisation and from the other might modify protein crowding and microviscosity. The distributed pattern of DT MRI abnormalities we found in the WM tracts clashes with the almost exclusive clinical involvement of the visual system in the majority of DOA patients and with the results of previous DT MRI studies of other hereditary optic neuropathies, such as LHON, which showed an almost selective involvement of

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the optic pathways [40, 41]. The reasons for the selective involvement of retinal ganglion cells in OPA1 mutation carriers are still unknown, even if different hypotheses have been considered, including high energetic requirements of these cells, particularly of the unmyelinated portion of their axons proximal to lamina crybrosa, as well as their higher exposition to permanent oxidative assault and pro-apoptotic stimuli [37, 39]. In addition, the different amount of OPA1 transcript levels in the retina vs the brain, testis, heart and skeletal muscle, which might lead to a cellspecific critical threshold of the amount of OPA1 protein, is likely to play a role [4]. The type of involvement of mitochondrial function (impairment of oxidative phosphorylation in the mitochondria in LHON vs deficits of mitochondrial fusion, cristae organisation and control of apoptosis in DOA) can also contribute to explain differences among the different forms of hereditary optic neuropathies. Remarkably, in our study no correlations were found between volumetric and diffusivity abnormalities quantified with MRI and clinical and neuro-ophthalmologic measures of disease severity. The lack of such an association agrees with current theories stating that these abnormalities may be present from birth or develop during the first decade of life [28, 42], as well as with the importance of OPA1 in embryonic development. Our study is not without limitations. First, since it is cross-sectional, future investigations should evaluate whether these abnormalities have a temporal evolution as well as the factors influencing the changes we observed. Second, despite the relatively large group of patients enrolled, they had heterogeneous patterns of OPA1 mutations, which might result in different protein variants. As suggested by the results of our comparison between patients with mutations causing haploinsufficiency vs those with missense mutations which point to more severe diffusivity abnormalities in the latter group, future studies should attempt to stratify patients according to the type of OPA1 mutation. Studies in asymptomatic OPA1 mutational carriers might also help to clarify more precisely the onset and nature of the structural abnormalities we detected as well as their role in the genesis of the clinical manifestations of the disease. Finally, considering the extensive involvement of WM areas we found with TBSS, future studies should also include a neuropsychological evaluation of these patients, to define whether such WM abnormalities are clinically relevant. Acknowledgements E.L. is supported by Pierfranco e Luisa Mariani Foundation, Italy. We thank Franco Carrara, Melissa Petrolini and Elisabetta Pagani for their contribution to the analysis of the data. Conflicts of interest of interest.

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The authors declare that they have no conflict

Ethical standard This study was performed in accordance with the ethical standards laid down in the 1965 Declaration of Helsinski and its later amendments.

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