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In this study, we used manual delineation of high-resolution magnetic resonance imaging (MRI) to determine the spatial and temporal characteristics of the cerebellar atrophy in spinocerebellar ataxia type 2 (SCA2). Ten subjects with SCA2 were compared to ten controls. The volume of the pons, the total cerebellum, and the individual cerebellar lobules were calculated via manual delineation of structural MRI. SCA2 showed substantial global atrophy of the cerebellum. Furthermore, the degeneration was lobule-specific, selectively affecting the anterior lobe, VI, Crus I, Crus II, VIII, uvula, corpus medullare, and pons, while sparing VIIB, tonsil/paraflocculus, flocculus, declive, tuber/folium, pyramis, and nodulus. The temporal characteristics differed in each cerebellar subregion: 1) Duration of disease: Crus I, VIIB, VIII, uvula, corpus medullare, pons, and the total cerebellar volume correlated with the duration of disease; 2) Age: VI, Crus II, and flocculus correlated with age in control subjects; 3) Clinical scores: VI, Crus I, VIIB, VIII, corpus medullare, pons, and the total cerebellar volume correlated with clinical scores in SCA2. No correlations were found with the age of onset. Our extrapolated volumes at the onset of symptoms suggest that neurodegeneration may be present even during the presymptomatic stages of disease. The spatial and temporal characteristics of the cerebellar degeneration in SCA2 are region-specific. Furthermore, our findings suggest the presence of presymptomatic atrophy and a possible developmental component to the mechanisms of pathogenesis underlying SCA2. Our findings further suggest that volumetric analysis may aid in the development of a non-invasive, quantitative biomarker.
Spinocerebellar ataxia type 2 (SCA2) is a familial autosomal dominant neurodegenerative disease characterized by poor control of gait, speech, coordination, and eye movements . It is associated with a CAG repeat expansion on chromosome 12q23-24.1  on the gene encoding for ataxin-2 , a protein that may be involved in RNA splicing  and may confer resistance to degeneration .
SCA2 shows progressive atrophy of the olivopontocerebellar system and the cerebral cortex . Functional changes have been shown to precede onset of overt clinical symptoms. Presymptomatic patients have been reported to show decreased saccade velocity . Presymptomatic patients have also shown decreased glucose metabolism through positron emission tomography in both the cerebellum and the pons . Global developmental impairment has been observed in SCA2 infants with high CAG repeat length (66 CAG repeats; typical adult-onset SCA2 has 34-57 CAG repeats) . The ortholog of human ataxin-2 plays an essential role in embryonic development of C.elegans .
Currently, investigating the region-specificity of the degeneration is complicated by individual anatomic variations and lack of automated algorithms that can reliably quantify lobule-specific atrophy. A few studies have previously examined in vivo the lobule-specific pattern of cerebellar atrophy in SCA2 [10,11] using voxel-based morphometry (VBM). However, the use of VBM with significantly atrophied brains is complicated by the challenges that arise during spatial normalization, including low sensitivity to cortical atrophy and distortion of macrostructures .
Our solution was to investigate the spatial and temporal characteristics of the cerebellar neurodegeneration in SCA2 through manual segmentation of regions of interest. Our previous findings have shown disease-specific anatomic differences in the pons and flocculus of the cerebellum [13,14]. Furthermore, we have shown that the atrophy of the brainstem and the total cerebellum correlates with the duration of disease and the severity of neurological deficits . This suggests MRI as a promising surrogate biomarker. In light of this, we used high-resolution MRI to characterize the differences in the spatial and temporal profile of neurodegeneration in the pons and different subregions of the cerebellum in SCA2.
All participants provided written informed consent according to a protocol approved by the Institutional Review Board of University of California, Los Angeles, School of Medicine. This protocol is in accordance with the declaration of Helsinki on ethical principles for medical research involving human subjects.
Eleven patients (10 females/1 male) with SCA2 (either genetically confirmed or with a genetically confirmed family history) and eleven neurologically normal age- and sex-matched controls were recruited for this study. The data from the single male subject with SCA2 and the single matched male control were excluded to prevent gender bias. Demographic and clinical information for patients and controls are displayed in Table 1.
All participants completed a medical questionnaire that evaluated past medical history, family medical history, progression of disease, overall health, and social history. The duration of disease was defined from their first self-reported symptoms of ataxia. Subjects were also assigned a Functional Staging score For Ataxia (FSFA) from 0 to 6 that emphasized mobility. The FSFA rating scale is a subset of the Unified Ataxia Disorders Rating Scale (UADRS), and was validated in a cohort study of Friedreich ataxia . The FSFA rating scale has also been shown to correlate with the morphometric changes of the pons and the total cerebellum in SCA2 .
Subjects were scanned on a 1.5T GE Signa scanner with a 3D-SPGR sequence using a sagittal slice orientation (TR=24ms, TE=4ms, slice thickness=1.2mm, FOV=240mm, matrix 256×256). Two scans from a single scanning session were co-registered, averaged, and reconstructed using the Brain Imaging Software Toolbox (Montreal Neurological Institute) to a 0.8×0.8×0.8 mm3 voxel size.
Regions of interest were manually delineated using Display software (Montreal Neurological Institute) by two blind raters. Intraclass correlation coefficients was determined (icc.m MATLAB Central File Exchange) (each rater delineated the same two controls and two SCA2s; 23 regions per patient [left and right hemispheric lobules, lobules of the vermis, and the total cerebellum]) to assess the interrater reliability.
Regions of interest were manually segmented based on Schmahmann's cerebellar atlas , which represents a modification of the Larsell classification (Figure 1a). The upper boundary of the pons was defined by an oblique plane connecting the most superior point of the pontine protrusion and the bottom of the inferior colliculi. The lower boundary of the pons was defined by a plane that originates from the bottom of the pontine protrusion, and is parallel to the upper boundary. The total cerebellar volume was defined to include the cerebellar cortex, arbor vitae, corpus medullare, and the deep cerebellar nuclei. The cerebellar cortex was subdivided into anterior lobe (lobules I-V), simplex (VI), Crus I, Crus II, paramedianus (VIIB), biventer (VIII), tonsil/paraflocculus (IX), and flocculus (X). The corpus medullare (central white matter and the deep cerebellar nuclei of the cerebellum) was defined to include the superior cerebellar peduncles up to the juncture with the brainstem and included part of the middle cerebellar peduncle, as defined by a plane orthogonal to the peduncle at the level of the flocculus. The boundary between corpus medullare and the hemispheres was defined by virtual lines connecting adjacent fissure bottoms. The vermis was then sub-divided into declive (VI), tuber/folium (VII), pyramis (VIII), uvula (IX), and nodulus (X). In the presence of cerebellar atrophy, the volumetric measurements excluded the cerebrospinal fluid space between the lobules of the cerebellum (Figure 1b).
The log-linear model of the regional volumes was computed to account for the exponentially decaying nature of neurodegeneration (rapid decrease in volume during the early stages of disease followed by a convergence to an asymptote). To directly assess the effects of ataxin-2 mutation, age-adjusted volumes were calculated for all regions using the rate of age-related neurodegeneration in the log(control volume) as the reference point for the regions that correlated with age. In order to evaluate localized disease-related atrophy, the log(age-adjusted volumes) of each regions of interest from SCA2 were compared to the regions of controls by using the Wilcoxon rank-sum test. The Bonferroni-Holm method was used to correct for effects of multiple comparisons.
In order to determine whether functional staging was specifically associated with morphometric changes in the pons and the subregions of the cerebellum, we compared FSFA scores, age of onset, duration of disease, and the number of CAG repeats with individual log(age-adjusted volumes) using Pearson's correlation analysis.
For regions that correlated with the duration of disease, we extended the log-linear model to project the relative hypothetical volume at the onset of symptoms (duration of disease = 0 year). The hypothetical volume at the onset of symptoms was then compared against the control volumes using the normalized cumulative distribution function score (normcdf.m, Matlab R2007a, statistics toolbox).
The mean intraclass correlation coefficient for interrater reliability was 0.991 (range: 0.9985-0.995; p < 0.0001).
The volumes of individual regions of interest are listed in Table 2. The volume of the anterior lobe (lobules I-V), lobule VI, Crus I, Crus II, VIII, uvula, corpus medullare, pons, and the total cerebellum was reduced compared to controls (p ≤ 0.05). In contrast, lobules VIIB, tonsil/paraflocculus, flocculus, declive, tuber/folium, pyramis, and nodulus were relatively spared (p > 0.05).
Consistent with our previous results , the age-adjusted volume of the pons (R2=0.768, p=0.001) and the total cerebellum (R2=0.775, p=0.001) correlated with the duration of disease. We additionally found Crus I (R2=0.815, p=0.0003), VIIB (R2=0.492, p=0.02), VIII (R2=0.527, p=0.02), corpus medullare (R2=0.733, p=0.002), and uvula (R2=0.670, p=0.004) to correlate with the duration of disease. Lobule VI (R2=0.374, p=0.06) and tonsil/paraflocculus trended towards significance (R2=0.336, p=0.08). Other regions did not reach statistical significance (p > 0.1).
Lobule VI (R2=0.438, p=0.04), Crus II (R2=0.678, p=0.003), and flocculus (R2=0.605, p=0.008) correlated with age in control subjects. Declive (R2=0.391, p=0.06) and the total cerebellum (R2=0.329, p=0.08) in controls trended towards correlating with age. In SCA2, declive (R2=0.415, p=0.04) correlated with age in SCA2, while tonsil/paraflocculus trended towards significance (R2=0.379, p=0.06).
Consistent with our previous results , the pons (Figure 2a; R2=0.856, p=0.0001) and the total cerebellum (Figure 2b; R2=0.720, p=0.002) correlated with FSFA. In addition, lobules VI (Figure 2c; R2=0.556, p=0.01), Crus I (Figure 2d; R2=0.554, p=0.01), VIIB (Figure 2e; R2=0.525, p=0.02), VIII (Figure 2f; R2=0.687, p=0.003), and corpus medullare (Figure 2g; R2=0.558, p=0.01) correlated with FSFA.
No correlations were found with the age of onset. The evaluation of CAG repeat length was excluded from the analyses due to the low number of patients with genetic diagnosis and the lack of variability in the number of CAG repeats.
Our projected regional volumes implied that the pons (−1.97σ; 2.5th percentile), corpus medullare (−2.14σ; 1.6th percentile), and the total cerebellum (−0.99σ; 16th percentile) would be smaller in SCA2 relative to controls even at the onset of symptoms (Figure 3). Crus I (−0.63σ; 26th percentile), VIII (−0.35σ; 36th percentile), and uvula (−0.71σ; 24th percentile) trended towards being smaller in size at the onset of symptoms relative to controls (Figure 3). Indeed, post-hoc qualitative analyses of the coronal images of the cerebellum from two subjects with the lowest duration of disease at the time of scan (duration of disease: 1 year and 6 years) confirmed that cerebellar atrophy was already qualitatively present at the early stages of SCA2 disease progression. Furthermore, the degeneration appeared to be localized to the anterior-superior regions while sparing the posterior-inferior regions of the cerebellum (Figure 4).
This is the first study to investigate the lobule-specific changes associated with SCA2 throughout the entire cerebellum via manual delineation of regions of interest. Our findings suggest that the different subregions of the pontocerebellar system are differentially vulnerable to the pathogenetic mechanisms of SCA2. More specifically, the volume of the anterior lobe, VI, Crus I, Crus II, VIII, uvula, corpus medullare, pons, and the total cerebellum was reduced compared to controls, while lobules VIIB, tonsil/paraflocculus, flocculus, declive, tuber/folium, pyramis, and nodulus were relatively spared. This spatial pattern of neurodegeneration is consistent with previous pathological findings [17,18] and our own previous partial evaluation of cerebellar subregions on MRI ; however, the current findings are in disagreement with previous VBM studies, which demonstrated the sparing of lobules I, II, Crus II, nodulus , VII and VIII . Presumably this is a reflection of the increased sensitivity to morphometric changes associated with manual delineation of regions of interest as well as the possible distortion of macrostructures during spatial normalization in VBM. Furthermore, our findings show that different cerebellar lobules may exhibit different temporal pattern of neurodegeneration. These morphologic changes of the pontocerebellar system may even begin years before the onset of clinical symptoms.
We found a disease-specific pattern of atrophy in SCA2 that differed from normal aging  and alcohol dependence . Our finding of increased atrophy of the cerebellar hemisphere relative to the vermis is also in contrast to the pattern of atrophy observed in spinocerebellar ataxia type 6 .
Our region-specific temporal pattern of neurodegeneration did not appear to be determined by the level of ataxin-2 expression alone. We found that the pons may be selectively more vulnerable to the effects of ataxin-2 during the early stages of disease relative to the cerebellar lobules. This does not correlate with the pattern of neurodegeneration expected from the differential level of regional ataxin-2 expression—only 55% of the pontine neurons express ataxin-2 , while 68.95% of the cells in the cerebellar hemisphere express ataxin-2 . Furthermore, we only found select lobules of the cerebellar hemisphere to be atrophied, despite the lack of difference in the level of ataxin-2 expression within the cerebellar hemispheres . It may be that the different histological and biochemical composition of these regions interact differently with ataxin-2exp. This may increase cellular vulnerability and decrease intrinsic neuroprotection in select regions of the cerebellum.
Projection of our data suggests that the neurodegeneration of the pontocerebellar system begins about 7.7 years before the onset of symptoms. Regional differences in size prior to the onset of clinical symptoms is not uncommon and has been reported in Alzheimer's disease  and Huntington's disease  in which regional atrophy predates the onset of clinical symptoms. A decrease in size of the brain prior to the onset of clinical symptoms has also been suggested in SCA7 .
The atrophy during the presymtomatic stage of the disease suggests a possible threshold in the level of neurodegeneration before symptoms appear. An alternative explanation is that patients were able to compensate for the initial deterioration of function, masking the symptoms caused by the early stages of neurodegeneration.
Alternatively, the decreased pontocerebellar volume may be a result of an impaired development. Recently it has been hypothesized that ataxin-2exp, but not the wild-type, sensitizes the inositol (1,4,5) triphosphate receptor type 1 (ITPR1) located on the membrane of the endoplasmic reticulum , and increases intracellular calcium release upon metabotropic glutamate signaling. Intracellular calcium signaling plays a crucial role in development . Indeed, ITPR1 knock-out has been shown to impair embryonic development in mice ; certainly, disruption of normal ITPR1 function via the binding of ataxin-2exp could also affect the balance between development and degeneration, and ultimately prevent normal growth.
There may be an effect of sampling bias due to the small number of subjects and because the final sample is composed exclusively of female subjects (despite gender-neutral recruitment practices). Additionally, SCA2 patients without genetic confirmation were included in this study on the basis of genetic confirmation of disease in their first degree family members. The chances of another diagnosis for these patients are low, but not zero. The study must be conducted in a larger and a more representative cohort to validate the spatial and temporal characteristics of the cerebellar degeneration in SCA2. The structural changes must also be examined longitudinally—including at-risk patients—with respect to the progression of disease. Due to the cross-sectional nature of our study, we could only infer the possibility of volumetric changes in pre-symptomatic SCA2 patients from our data. Tracking the longitudinal progression of cerebellar development and the course of atrophy will further advance our understanding of the effect of disease-specific mechanisms on the balance between development and presymptomatic atrophy. An alternate possibility is that there is a higher order pattern of degeneration in these regions, with very rapid degeneration in the first few years of disease following normal size at the onset of symptoms. If true, this would identify a critical period for intervention. Lastly, we could only infer the death of Purkinje cells via volumetric loss; future studies are needed to correlate volumetric loss with histopathology of the cerebellum.
We examined the spatial and temporal characteristics of the cerebellar neurodegeneration in SCA2. Our findings indicate that the atrophy is not uniform throughout the pontocerebellar structure, but is region-specific, selectively affecting the anterior lobe, lobule VI, Crus I, Crus II, VIII, uvula, corpus medullare, and pons while sparing lobules VIIB, tonsil/paraflocculus, flocculus, declive, tuber/folium, pyramis, and nodulus. The temporal characteristics of the cerebellar neurodegeneration in SCA2 are also region-specific. The neurodegeneration may precede the onset of symptoms by years. This may be a result of presymptomatic atrophy or developmental impairment or a combination of the two. Longitudinal volumetric MRI analyses of the cerebellar degeneration could provide us with insight into the time course of the mechanism underlying the pathogenesis of SCA2. Understanding the region-specificity and the temporal characteristics of the neurodegeneration will help in the treatment, diagnosis, prognosis, and the staging of disease.
This work was supported by the Arnold-Chiari Foundation; the Robin Zee Fund; the Dana Foundation Program for Brain and Immuno-Imaging; the Research to Prevent Blindness Core Grant; and the National Institutes of Health [grant numbers 1K23EY015802, 5T32DC00023, 5T32MH019950, 5T32GM007057, R01 EY01849, 1R01NS056307, R01NS054255, 5RC1NS068897, 5R01EY019347 and 5R21NS059830]. We would also like to thank Elizabeth Murray for her technical assistance.
Conflict of Interest: There is no financial interest to disclose.