Cerebellum DOI 10.1007/s12311-014-0574-3

ORIGINAL PAPER

Comprehensive Study of Early Features in Spinocerebellar Ataxia 2: Delineating the Prodromal Stage of the Disease Luis Velázquez-Pérez & Roberto Rodríguez-Labrada & Edilia M. Cruz-Rivas & Juan Fernández-Ruiz & Israel Vaca-Palomares & Jandy Lilia-Campins & Bulmaro Cisneros & Arnoy Peña-Acosta & Yaimeé Vázquez-Mojena & Rosalinda Diaz & Jonathan J. Magaña-Aguirre & Tania Cruz-Mariño & Annelié Estupiñán-Rodríguez & José M. Laffita-Mesa & Rigoberto González-Piña & Nalia Canales-Ochoa & Yanetza González-Zaldivar

# Springer Science+Business Media New York 2014

Abstract The prodromal phase of spinocerebellar ataxias (SCAs) has not been systematically studied. Main findings come from a homogeneous SCA type 2 (SCA2) population living in Cuba. The aim of this study was to characterize extensively the prodromal phase of SCA2 by several approaches. Thirty-seven non-ataxic SCA2 mutation carriers and its age- and sex-matched controls underwent clinical assessments, including standardized neurological exam, structured interviews and clinical scales, and looking for somatic and autonomic features, as well as a neuropsychological battery, antisaccadic recordings, and MRI scans. Main clinical L. Velázquez-Pérez (*) : R. Rodríguez-Labrada : A. Peña-Acosta : Y. Vázquez-Mojena : T. Cruz-Mariño : A. Estupiñán-Rodríguez : J. M. Laffita-Mesa : N. Canales-Ochoa : Y. González-Zaldivar Center for the Research and Rehabilitation of Hereditary Ataxias, Libertad Street 26, Holguín 80100, Cuba e-mail: [email protected] E. M. Cruz-Rivas : J. Lilia-Campins Clinical-Surgical Hospital “Lucía Iñiguez”, Holguín, Cuba J. Fernández-Ruiz : I. Vaca-Palomares : R. Diaz Neuropsychology Laboratory, Physiology Department, Medicine School, UNAM, Mexico City, Mexico B. Cisneros Department of Genetics and Molecular Biology, CINVESTAV-IPN, Mexico City, Mexico J. J. Magaña-Aguirre Laboratory of Genomic Medicine, Department of Genetics, INR, Mexico City, Mexico R. González-Piña Department of Brain Plasticity, National Rehabilitation Institute (INR), Mexico City, Mexico

somatic features of non-ataxic mutation carriers were cramps, sensory symptoms, sleep disorders, and hyperreflexia, whereas predominating autonomic symptoms were pollakiuria/ nocturia, constipation, and frequent throat clearing. Cognitive impairments included early deficits of executive functions and visual memory, suggesting the involvement of cerebro-cerebellar-cerebral loops and/or reduced cholinergic basal forebrain input to the cortex. Antisaccadic task revealed impaired oculomotor inhibitory control but preserved ability for error correction. Cognitive and antisaccadic deficits were higher as carriers were closer to the estimated onset of ataxia, whereas higher Scale for the Assessment and Rating of Ataxia (SARA) scores were associated most notably to vermis atrophy. The recognition of early features of SCA2 offers novel insights into the prodromal phase and physiopathological base of the disease, allowing the assessment of its progression and the efficacy of treatments, in particular at early phases when therapeutical options should be most effective. Keywords SCA2 . Presymptomatic . Prodromal phase . Biomarkers . Cognitive disorders . Antisaccadic

Introduction Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant cerebellar ataxia caused by the abnormal expansion of CAG triplet repetitions on the coding region of ATXN2 gene (12q24.1). This mutation, in turn, causes the expression of an expanded polyglutamine tract in the ataxin 2 protein. The clinical picture of SCA2 includes a progressive cerebellar syndrome accompanied by saccadic slowing, peripheral neuropathy, autonomic dysfunctions, sleep

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disturbances, cognitive abnormalities, and signs of motor neuron involvement [1]. Cuba has the highest SCA2 prevalence in the world, where a founder effect has been found in the Holguin province. On the island, there are 163 affected families encompassing almost 600 SCA2 living patients and almost 8,000 at-risk individuals. Within this latter group, 2,060 subjects are SCA2 patients’ first-degree relatives. This results in an estimated ATXN2 mutation prevalence of near 28.51 cases per 105 inhabitants in the whole country and 182.75 cases per 105 inhabitants in the Holguin province [1, 2]. Most of the SCA2 phenotypic studies have been focused on the clinical stages, emphasizing the natural history of the disease and the progression patterns of the cerebellar syndrome, as well as the non-cerebellar features [3–5]. Nevertheless, the prodromal phase of the disease has not been systematically studied, which hinders the design of early therapeutic options, useful when the neurodegeneration is not so advanced. The most detailed characterization of prodromal SCA2 comes from the large and homogeneous population of Cuban non-ataxic SCA2 mutation carriers. This characterization was based on the application of an 11-year presymptomatic diagnostic program that included the molecular diagnosis of more than 1,000 at-risk individuals [6]. This diagnostic program also included several clinical and electrophysiological assessments in at-risk individuals that have resulted in the identification of multiple prodromal features and the development of sensitive preclinical biomarkers [7–11]. Recently, a large follow-up study initiated in 40 at-risk individuals for SCA2 demonstrated the progressive nature of some early features, such as the muscle cramps, sensory symptoms, abnormal deep tendon reflexes, and subtle coordination deficits, and confirmed the correlation between the mutation size and the onset of some of these early non-motor features [12]. Other relevant studies aimed to characterize the preclinical features of SCA2 include an interview-based retrospective analysis in 97 European SCA2 patients who reported sleep disorders, problems to hand writing, and dysarthria as significantly specific early symptoms [13]. Besides, a prospective study with 31 non-ataxic SCA2 mutation carriers enrolled from 14 distinct European centers reported mild coordination deficits and muscle cramps [14], similar to previous reports from the Cuban SCA2 non-ataxic cohort [8, 12]. Nevertheless, other phenotypic features, such as cognitive decline and antisaccadic performance, have not been investigated in SCA mutation carriers. Therefore, to gain more insights into the prodromal phase of SCA2, we developed a comprehensive study including somatic, autonomic, cognitive, antisaccadic, and imaging assessments in 37 Cuban non-ataxic SCA2 mutation carriers and their sex- and agematched non-SCA2 mutation carriers.

Methods Subjects Sixty non-ataxic relatives of SCA2 patients were assessed for eligibility to participate in the present study in the Center for the Research and Rehabilitation of Hereditary Ataxias in Holguín. To be included, the non-ataxic relatives had to meet the following criteria: (a) absence of definite cerebellar syndrome, (b) CAG expansion above 32 repeats in the ATXN2 gene, and (c) age between 18 and 70 years. Exclusion criteria for participation were chronic diseases affecting the nervous system, psychiatric disorders, chronic alcohol abuse, and pregnancy. Among the 60 eligible nonataxic relatives, 23 were excluded on the basis of inclusion/ exclusion criteria (12 cases) or by declination to participate (11 cases). Therefore, the present study enrolled 37 nonataxic SCA2 mutation carriers (24 female) with ages ranging from 21 to 70 years (mean 40.08; standard deviation (SD)± 11.68), probable age of onset from 36 to 69 years (mean 54.19; SD±11.77), and CAG repeat sizes from 32 to 39 repetitions (mean 35.65; SD±2.03). The probable age at onset was estimated from each CAG repeat length, between 34 and 39 repeats, using a survival analyses model obtained in a large population of 924 Cuban SCA2 patients and nonataxic carriers [15]. Thirty-seven healthy subjects belonging to no-SCA2 related families (24 females, ages from 23 to 71 years, mean 41.0; SD 11.9) were randomly chosen as age- and sex-matched controls. The study was approved by the Ethics Committee of the Center for the Research and Rehabilitation of Hereditary Ataxias (Holguín, Cuba) and was conducted according to the Declaration of Helsinki. Each subject gave written informed consent for participation in the study. As the non-ataxic SCA2 mutation carriers proceeded from the Cuban Program of Presymptomatic Diagnosis [6], they and most investigators were not blind to the carrier status of the relatives. Nevertheless, to diminish the bias from known diagnosis, the study participants (both non-ataxic SCA2 mutation carriers and controls) underwent the same clinical, neuropsychological, antisaccadic, and imaging assessments, following standardized and validated procedures, which allow the phenotypical analysis of carriers and controls through a relatively equivalent manner. Anyway, the effect of this bias seems not to be significant for non-ataxic SCA2 mutation carriers, since recent findings showed that awareness of the carrier status has no influence on most clinical assessments included in the present paper [14]. Neurological Exam and Clinical Scales All subjects were clinically evaluated following the standardized Mayo Clinic procedures for neurological exam [16] and

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structured medical interview. The evaluation of cerebellar features was performed using the Scale for the Assessment and Rating of Ataxia (SARA) [17], whereas the noncerebellar features were assessed with the Inventory of Nonataxia Symptoms (INAS) scale [5]. Autonomic symptoms were evaluated with the Scales for Outcomes in Parkinson’s Disease-Autonomic (SCOPA-AUT) scale [18]. Neuropsychological Assessment INECO Frontal Screening Test [19] It is a standardized battery of specific tests to assess executive functions. This Spanish-validated battery consists on nine subtests (motor programming, conflicting instructions, go/no go task, backward digit span, verbal working memory, spatial working memory, abstraction capacity, and verbal inhibitory control) and yields a total score of 30 points. Scores below or equal to 25 points are considered as abnormal. Stroop Color-Word Interference [20] This language-free test is designed to assess ability to suppress interfering stimuli and the selective attention. It consists of a color-naming condition, where subjects name the color of colored patches, and an interference condition where subjects are shown an array of color names printed in different colored inks. The analyzed parameters were the time to complete this task, obtained by subtracting the time needed for the color-naming condition from the time needed for the interference condition and the number of interference errors. Stroop interference times and interference errors above the 2 SD thresholds of healthy controls’ mean scores were considered as abnormal. Phonemic Verbal Fluency Test [21] This test measures the ability to access to verbal storage systems and monitoring of retrieved information. All subjects were asked to name as many items as possible from phonemic category (nouns starting with the letters F, A, and S) during 1 min for each letter. Mean successful nouns were analyzed. Abnormal scores were considered for the number of nouns below the 2 SD threshold of healthy controls’ mean score. Evoked Verbal Memory Test [22] For measures of verbal memory, all subjects were tested for the immediate and delayed recall of ten-item uncategorized word list. Subjects were asked to recall each list immediately after presentation and after a delay of 20 min. For immediate recall, a maximum of ten trials were applied. The analyzed parameters were the recalled words in the first trial, the number of trials needed to recall the entire list, and the recalled words in the delayed trial. Abnormal scores for the recalled words in the first and delayed trials were considered if the number of words were less than the 2 SD threshold of healthy controls’ mean score, whereas for the number of trials needed to recall the entire list,

it was considered for trials above the 2 SD threshold of healthy controls’ mean score. Cambridge Neuropsychological Test Automated Battery Two subtests from the Cambridge Neuropsychological Test Automated Battery (CANTAB) [23] were administered via computer. Participants underwent various training trials to learn the requirements of each task, and responses were recorded directly with a touch-sensitive screen. All participants were able to sustain attention and comply with task demands. Intradimensional/Extradimensional Set Shift (IED) Test [24] This task is a computerized analog of the Wisconsin Card Sorting Test and measures the ability to attend to specific attributes of compound stimuli, shifting attention from one attribute to another when required. Participants are presented with a series of multidimensional stimuli consisting of shapes and lines and must learn through trial and error to respond selectively to one specific shape, ignoring the other shape and the lines. Feedback teaches the participant which stimulus is correct, and after six correct responses, the stimuli and/or rules are changed. These shifts are initially intradimensional (e.g., color-filled shapes remain the only relevant dimension), then later extradimensional (white lines become the only relevant dimension). Participants progress through the test by satisfying a set criterion of learning at each stage (six consecutive correct responses). If at any stage the participant fails to reach this criterion after 50 trials, the test terminates. The outcome measure was the number of errors preceding extradimensional sets (EDS). Number of errors above the 2 SD threshold of healthy controls’ mean errors were considered as abnormal. Delayed Matching to Sample (DMS) Tests [24] This test assesses forced choice recognition memory for novel nonverbalizable patterns and tests both simultaneous and shortterm visual memory. Participants are shown a complex visual pattern (the sample) and then, after a brief delay, four similar patterns. The participant must touch the pattern which exactly matches the sample. The outcome measure for this task was the numbers of correct responses. Abnormal scores were considered when the number of correct responses was less than the 2 SD threshold of healthy controls’ mean score. Antisaccadic Eye Movement Assessment Eye movements were recorded binocularly with a twochannel Otoscreen AC electronystagmograph (JaegerToennies, Germany), using silver-silver chloride electrodes over the right and left outer canthus of the eyes. The signal was amplified and filtered (band-pass filter: 0.2–70 Hz). The data were sampled at a frequency of 200 Hz with a time base

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of 1,000 ms/division, sensitivity of 200 μV/division, and a time constant of 8 s. Antisaccade task was applied in two sets of 22 trials. The first set was administered to familiarize the subjects with the task and was excluded from the analyses. Each trial started with the appearance of a central fixation stimulus during 3 s (intertrial interval). After this period time, the central stimulus was extinguished and a peripheral stimulus appeared at 30° predictable amplitude, to the left and to the right of the central fixation stimulus, alternatively. One second later, the peripheral stimulus disappeared and the central fixation stimulus reappeared beginning the subsequent trial. Subjects were instructed to fixate the target in the central position and then to redirect their gaze to the mirror image location of the target as soon as it appeared in the periphery. Antisaccadic (AS) task was analyzed according validated methodological issues [25] and included the following measures: (i) inhibition AS error rate, percentage of trials in which subjects initiate an eye movements toward the target; (ii) percentage of AS correction, percentage of inhibition AS errors that were spontaneously corrected by a corrective eye movement contrary to the target direction; (iii) latency of correct AS, time interval from the appearance of the eccentric target to the onset of the correct antisaccadic eye movement; and (iv) latency for AS error correction, time interval it took the subject to initiate the corrective antisaccade from the last position of the eye in the error saccade direction. Magnetic Resonance Imaging For MRI procedures, a subgroup of 24 non-ataxic SCA2 mutation carriers and its controls were scanned on 0.23-T scanner (Philips, Tokyo, Japan). The imaging protocol comprised a sagital T2-weighted spin echo dataset measured with a standard head coil. The section thickness was 5 mm, repetition time (TR) 24 ms, and echo time (TE) 9 ms. All MRI data were analyzed by an investigator blinded to the subjects diagnosis, using the image analysis software Imagis v1.3 (Cuba, 2002). Regions of interest were manually delineated based on a cerebellar atlas [26]. Five parameters were measured 1. Area of the posterior cranial fossa (defined anteriorly, by the posterior border of the sphenoid bone, superiorly for the line between the posterior clinoid and the anterior part of inferior border of the straight sinus, posteriorly by the inferior border of the straight sinus and the border of occipital bone, and inferiorly for the line between the inferior points of occipital and sphenoid bones) 2. Area of the cerebellar vermis 3. Area of the cerebellar hemispheres 4. Maximal antero-posterior diameter of the pons (perpendicularly to the pons axis) 5. Maximal rostro-caudal diameter of the pons

Molecular Genetics Measures DNA extracted from peripheral venous blood underwent the amplification of CAG-rich region in SCA2 gene by PCR and determination of mutation size by polyacrylamide gel electrophoresis. Statistical Analysis Group comparisons were made with Student’s t test (two tailed), followed by the Bonferroni adjustment test. Frequencies of preclinical symptoms and signs were compared using a Chi-square test, whereas correlation analyses were performed by Pearson product moment correlation tests. An arbitrary level of 5 % statistical significance was assumed. These analyses were conducted using the STATISTICA software package (StatSoft, Inc., 2003 STATISTICA data analysis software system, version 6. www.statsoft.com).

Results Clinical Somatic Assessments The main clinical somatic features of SCA2 mutation carriers, in comparison with healthy controls, are shown in Fig. 1. The most prominent complaint among the SCA2 mutation carriers, was the presence of painful muscle cramps that occur during wakefulness or sleep times (reported by 81.08 % of them). Other important manifestations were the sensory neuropathic symptoms (62.16 %), which included paresthesia, hypoesthesia, and hypopallesthesia. Hyperreflexia was detected in 43.24 % of non-ataxic SCA2 mutation carriers. Also, these subjects disclosed a reduced subjective sleep quality based on higher prevalence of insomnia and nocturnal muscle cramps (48.65 %); nevertheless, history of restless leg syndrome, REM sleep behavior disorder, parasomnias, and other sleep complaints were not frequent. Also, bedside examination revealed oculomotor disturbances in 21.62 % of cases. Slight slowing of horizontal saccades was observed in 16.21 %, whereas horizontal gaze-evoked nystagmus was only present in 8.34 % of the SCA2 carriers. SARA scores of non-ataxic SCA2 mutation carriers ranged from 0 to 4 points (mean±SD: 1.07±1.42). Twenty-five (67.6 %) carriers had a score of 0, whereas positive scores were mainly obtained in items referring heel-shin slide (21.6 %), fast alternating hand movements (18.9 %), and gait (16.2 %), but, in no case with positive scores, the punctuations were above 1. Therefore, the gait disturbances, the main criteria to define the disease onset [13], were not permanent and only discernible by the tandem task. SARA scores were 0 for all healthy controls. No correlations between SARA score

Cerebellum Fig. 1 Somatic features preceding cerebellar syndrome in non-ataxic SCA2 mutation carriers. p>0.05: non-significant; p<0.05: significant

with age (r=0.04; p=0.781), CAG repeats (r=0.07; p= 0.676), or time to ataxia onset (r=−0.23; p=0.218) were observed. Regarding INAS scale, non-ataxic SCA2 mutation carriers showed scores between 0 and 5 points with a mean of 1.85±1.44. INAS score was not correlated to age (r=0.02; p= 0.869), CAG repeats (r=−0.13; p=0.484), and time to ataxia onset (r=−0.01; p=0.997); however, this score was correlated to SARA (r=0.38; p=0.003). Clinical Autonomic Assessments Non-ataxic SCA2 mutation carriers had higher SCOPA-AUT scores compared to healthy controls (carriers: 5.89±5.68; controls: 3.04±4.39; p=0.04). The most affected autonomic functions were gastrointestinal and urinary functions (Fig. 2), whereas predominating symptoms were pollakiuria (57.14 %), nocturia (42.85 %), constipation (24.91 %), and frequent throat clearing (21.42 %). SCOPA-AUT indices showed no correlation with neither age (r=0.02; p=0.889), CAG repeats (r=−0.01; p=0.971), time to ataxia onset (r= −0.30; p=0.188), nor SARA score (r=0.10; p=0.499). Fig. 2 Percentage of autonomic dysfunctions in non-ataxic SCA2 mutation carriers compared to controls. p>0.05: non-significant; p<0.05: significant

Cognitive Dysfunctions Results for cognitive dysfunctions are shown in Table 1. Mean score of INECO test was significantly reduced in non-ataxic SCA2 mutation carriers, compared to that in controls (p<0.001). INECO scores below 25 were obtained in 14 carriers (37.8 %). Additionally, an increase of the time for the Stroop interference task was observed in the carriers (p=0.021), but the number of interference errors was not statistically different to that in controls (p=0.073). Phonemic verbal fluency test disclosed a decrease of correct nouns (p= 0.045). Twelve (32.4 %) and seven (18.9 %) out of all nonataxic SCA2 mutation carriers had abnormally prolonged times for the Stroop interference task and reduced scores for verbal fluency, respectively. Verbal memory was normal even for immediate and delayed trials. Analysis of CANTAB tasks in non-ataxic SCA2 mutation carriers revealed significant increase of errors in the IED task when an extra dimensional shift preceded the trial (p=0.009). Also, in the DMS task, a marked reduction of correct responses was observed (p=0.017). Abnormal performances for IED and

Cerebellum Table 1 Performance on neuropsychological tests of non-ataxic SCA2 mutation carriers and healthy controls

p<0.05: significant; p>0.05: nonsignificant SD standard deviation

Neuropsychological tests

Pre-SCA2 (mean±SD)

Controls (mean±SD)

p

INECO test Interference Stroop test Time (s) Errors Phonemic fluency test

20.35±4.96

24.06±2.35

<0.001

56.33±23.43 2.52±2.77

41.24±13.70 1.12±1.54

0.021 0.073

10.25±2.81

0.045

6.29±1.36 5.00±1.37 8.35±1.58

0.278 0.584 0.518

6.59±2.62

0.009

34.03±3.30

0.017

Successful words 8.47±2.48 Verbal memory test Recalled words in 1st attempt 5.81±1.29 Number of attempts 5.33±2.15 Recalled words delayed trial 8.05±1.28 Intradimensional/extradimensional set shift (IED) Errors (EDS preceding) 9.42±5.22 Delayed matching to sample (DMS) Correct responses 31.42±4.66

DMS tasks were observed in 9 (24.3 %) and 13 (35.1 %) non-ataxic SCA2 mutation carriers, respectively. INECO score was significantly correlated with time to ataxia onset (r=0.45; p=0.027) but was not correlated to age (r=−0.17; p=0.220), CAG repeats (r=−0.06; p=0.771), nor SARA score (r=−0.25; p=0.856). Regarding the CANTAB variables, the number of IED errors with preceding EDS and the DMS correct responses correlated with age (r=0.48; p= 0.017 and r=−0.56; p=0.004, respectively) and time to ataxia onset (r=−0.51; p=0.016, Fig. 3a, and r=0.46; p=0.044, Fig. 3b, respectively) in the non-ataxic SCA2 mutation carriers. In the case of control subjects, the DMS correct responses were correlated to age (r=−0.30; p=0.034, Fig. 3d), but the IED errors with preceding EDS were uncorrelated (r= −0.03; p=0.853, Fig. 3c).

mutation carriers and healthy controls is shown in Fig. 5. No significant differences were detected for the area of the posterior cranial fossa. Nevertheless, the areas of the cerebellar vermis and cerebellar hemispheres as well as the anteroposterior and rostro-caudal diameters of the pons were notably reduced in non-ataxic SCA2 mutation carriers. Correlation analyses disclosed an inverse association between the area of cerebellar vermis and the SARA score (r=−0.70; p=0.016), but no other MRI parameters showed correlation with any demographical, clinical, and molecular variables (Table 2).

Antisaccadic Abnormalities

Here, we set out to do a comprehensive study of prodromal manifestations in non-ataxic SCA2 mutation carriers. The present results revealed the existence of multiple clinical alterations as well as cerebellar, pontine, and cortical brain atrophy that preceded the onset of ataxia for many years, characterizing the prodromal stage of SCA2. Among the clinical manifestations described in this paper, the most novel features were the cognitive dysfunctions, assessed by neuropsychological tests and antisaccadic task, and the early dysautonomic abnormalities.

Antisaccade assessment in non-ataxic SCA2 mutation carriers disclosed a significant increase of inhibition AS error rate, compared to controls, but for the percentage of AS correction, there were no statistical differences among groups (Fig. 4a). Moreover, the carrier group showed prolonged latencies for correct AS and for AS error correction (Fig. 4b). Both AS latencies were inversely correlated to time for ataxia onset (latency of correct AS: r=−0.55; p=0.006; latency for AS error correction: r=−0.69; p=0.001).

Discussion

Early Clinical Somatic Features Brain MRI Study MRI studies in non-ataxic SCA2 mutation carriers revealed light cerebellar atrophy in 15 out of 24 assessed individuals (62.5 %), followed by pons atrophy in 7 out of 24 (29.2 %) and light atrophy of the frontal cortex, in 5 cases (20.8 %). Means comparison for MRI findings between non-SCA2

Among the early features observed in non-ataxic SCA2 mutation carriers, muscle cramps and sensory symptoms were the most frequent complaints, as was previously demonstrated by a large follow-up assessment of prodromal markers in a distinct cohort of Cuban non-ataxic SCA2 mutation carriers [12]. Both features reveal the early involvement of peripheral

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Fig. 3 Influence of time to ataxia onset on IED errors preceding EDS (a) and DMS correct responses (b) in non-ataxic SCA2 mutation carriers as well as influence of age on IED errors preceding EDS (c) and DMS

correct responses (d) in controls. IED intradimensional/extradimensional set shift, EDS extradimensional sets, DMS delayed matching to sample. p>0.05: non-significant; p<0.05: significant

nerves in SCA2, so muscle cramps are suggestive of early involvement (hyperexcitability) of motoneurons, especially at intramuscular nerve terminals [27], whereas sensory symptoms result from the loss of cells in the dorsal root ganglia [28]. Early hyperreflexia in non-ataxic SCA2 mutation carriers suggests loss or dysfunction of corticospinal projections [29], which could be anatomopathologically supported by the early loss of giant Betz pyramidal cells in the primary motor cortex [30].

Findings on sleep and oculomotor disorders were consistent with previous video-polysomnographical and electronystagmographical recordings obtained in other cohorts of Cuban non-ataxic SCA2 mutation carriers, respectively. These findings suggest an early dysfunction or neurodegeneration of pontine regions involved on sleep regulation and saccade generation [10, 11], which is compatible with our imaging data and previous papers reporting the pontine atrophy in non-ataxic SCA2 mutation carriers [14, 31].

Fig. 4 Antisaccade performance in non-ataxic SCA2 mutation carriers. a Inhibition antisaccade error rates. b Antisaccade latencies. Error bars represent standard error of the means. p>0.05: non-significant; p<0.05: significant

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Fig. 5 MRI findings of non-ataxia SCA2 mutation carriers and healthy controls. Little boxes represent the mean, bigger boxes represent the mean± standard error (SE), and whiskers represent the means±1.96 SE. p>0.05: non-significant; p<0.05: significant

Early Clinical Autonomic Features Autonomic dysfunctions have been reported in patients with SCAs [32–34], but their prevalence in prodromal stages has not been systematically studied [35], so the present findings represent the most thorough clinical characterization of the autonomic nervous system in this disease stage. The clinical assessment of autonomic symptoms in non-ataxic SCA2 mutation carriers using the validated SCOPA-AUT scale revealed a wide range of dysautonomic features preceding the ataxia onset, which

demonstrated the usefulness of this clinical non-cerebellar scale to detect early symptoms of the disease since early stages. Pollakiuria and nocturia were the most important autonomic complaints for non-ataxic SCA2 mutation carriers, indicating an early dysfunction of the autonomic and somatic nervous control of the bladder [36]. Some studies in animal models [37] and humans [38] have suggested that these symptoms may result from the parasympathetic denervation and subsequent reinnervation of denervated cells by excitatory sympathetic preganglionic pathways, leading to bladder overactivity.

Table 2 Correlation of MRI parameters with demographical, molecular, and clinical variables in non-ataxic SCA2 mutation carriers MRI parameters

Area of the posterior cranial fossa Cerebellar areas Vermis Hemispheresa Pontine diameters Antero-posterior Rostro-caudal

Correlation coefficients (R) with Age

Expanded CAG

Time to ataxia onset

SARA score

INAS

INECO

SCOPA

−0.28, ns

0.16, ns

0.20, ns

0.13, ns

−0.40, ns

0.10, ns

0.04, ns

0.25, ns 0.26, ns

−0.22, ns −0.26, ns

0.13, ns 0.06, ns

−0.70* −0.25, ns

−0.06, ns 0.01, ns

−0.18, ns 0.38, ns

0.36, ns −0.15, ns

0.04, ns 0.21, ns

0.05, ns −0.16, ns

0.24, ns 0.05, ns

−0.37, ns 0.10, ns

−0.15, ns 0.25, ns

−0.07, ns 0.07, ns

0.34, ns −0.35, ns

ns not significant, SARA Scale for the Assessment and Rating of Ataxia, INAS Inventory of Non-ataxia Symptoms, SCOPA Scales for Outcomes in Parkinson’s Disease-Autonomic *

p<0.05

a

The mean of both cerebellar hemispheres is shown

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Besides, these conditions may result from damage to somatic descending inhibitory pathways arising from higher brain centers, such as the prefrontal cortex, anterior cingulate gyrus, and the pontine micturition centers [39]. Constipation in non-ataxic SCA2 mutation carriers could result from the loss of parasympathetic fibers proceeding from the dorsal motor nucleus of the vagus, the main responsible for gastrointestinal motility [40, 41]. In fact, an early neuropathological study in six SCA2 patients has revealed a moderate to severe cell loss and astrogliosis in this brain stem autonomic nucleus as well as atrophy and myelin loss in the vagal nerve, even for a case with only 5 years of disease duration [42]. Finally, the frequent throat clearing can be interpreted as an early dysphagia [13] and could result from subtle impairments of the ingestive process, which involves multiple autonomic nuclei that undergo degeneration in SCA2, such as the medial, pigmented, and commissural solitary nuclei, as well as the ambiguus and dorsal motor vagal nuclei [43]. Also, the neuronal loss of trigeminal, facial, and hypoglossal nuclei in SCA2 could contribute to the frequent throat clearing, since these nuclei are involved in most phases of the swallowing [44, 45]. Indeed, this physiopathological approach is supported by early electrophysiological findings showing signs of myelin loss in trigeminal, facial, and hypoglossal nuclei from a distinct cohort of Cuban non-ataxic SCA2 mutation carriers [44].

CANTAB-derived outcomes could also be excluded, since the analyzed parameters were not timed and depend on making correct decisions rather than motor precisions. Executive and memory disturbances preceding the cerebellar syndrome in SCA2 could be considered as early markers of the neurodegenerative process in the frontal and temporal cortices, basal ganglia, cerebellum, and other regions involved in these cognitive functions. Specifically, the executive deficits of non-ataxic SCA2 mutation carriers suggest the early degeneration of specific prefrontal regions and/ or the involvement of fronto-ponto-cerebellar and frontobasal pathways [46]. Moreover, the visual memory deficits could be explained by the involvement of medial temporal lobe, since abnormal DMS performances have been proposed to be primarily sensitive to damage on this brain area [47]. Furthermore, the reduced cholinergic basal forebrain input to the frontal or temporal cortex could be proposed as putative mechanism underlying cognitive deficits in prodromal SCA2 [48]. A key finding of this paper was the significant associations between the performance of INECO test and both CANTABderived tasks and the time to ataxia onset, which suggests the progressive nature of cognitive dysfunctions even in early stages of the disease and identify these cognitive parameters as predictive biomarkers of the disease.

Early Cognitive Disorders

Antisaccadic Deficits in Prodromal SCA2

To our knowledge, these are the first results showing cognitive decline before the ataxia onset in any SCAs. The reduced INECO score and altered performances of Stroop, phonemic fluency, and IED tests suggest the presence of early executive dysfunctions, whereas the abnormal performance on DMS task suggests early impairments of visual memory in nonataxic SCA2 mutation carriers. Among the subcomponents of the executive functions, the most discernible deficits in prodromal SCA2 were the reduced inhibitory control and set shifting assessed by the Stroop [20] and IED test [24], respectively, as well as the impaired monitoring of retrieved information and access to verbal storage systems assessed by the verbal fluency test [21]. The impaired performances in the aforementioned neuropsychological tests also revealed an early deficit of the working memory systems and sustained attention in SCA2, two important functions that mediate temporary storage and manipulation of the information necessary for carrying out complex cognitive tasks [45]. Given that the Stroop and verbal fluency tests imply verbal responses [20, 21], their performances could be affected by speech disturbances. Nevertheless, since no mutation carriers had dysarthria problems (all of them had 0 points in the SARA scale’s speech subitem), we exclude its effect as important determinant for the outcomes of both tasks. Similarly, the effect of subtle coordinative deficits of some carriers on

The antisaccade task has been usually considered as an objective method to assess executive functions (inhibitory control, working memory, and sustained attention), due to its easy quantification, reliability, and high correlation with neuropsychological tests [49]. Nevertheless it has not been used systematically in SCAs as a diagnostic or research tool and never had been studied in non-ataxic mutations carriers [50], which places the present work as pioneer reporting antisaccade deficits preceding cerebellar syndrome in SCAs. The neural circuitry underlying antisaccades includes the prefrontal cortex, superior colliculus, striatum, cerebellum, and other cortical and subcortical areas [49]. Thus, according to the available neuroanatomical data of SCA2 [28, 30, 31], it can be inferred that the pattern of antisaccadic deficits in nonataxic SCA2 mutation carriers is caused by the early involvement of frontal regions and/or cortico-cerebellar loops [51]. As a relevant finding, the time to initiate a successful AS and the time to correct AS errors showed a significant association to the time before ataxia, so non-ataxic carriers with higher probabilities to be clinically diagnosed with the cerebellar syndrome had worst antisaccadic performance. This finding suggests that antisaccadic abnormalities progress insidiously during the prodromal stage of SCA2, identifying these variables as preclinical biomarkers of the disease. Besides, given that antisaccade task provides objective

Cerebellum

hands- and language-free measures for executive dysfunctions [49], it can be considered as a better cognitive biomarker than neuropsychological tests, having a higher usefulness for the diagnosis and prognosis of the disease since prodromal stages. Furthermore, compared to saccadic slowing, as another SCA2 prodromal feature assessed by electronystagmography [11], the antisaccadic abnormalities seem to be more useful in the clinical practice during early disease stages, as these in addition to estimating the probabilities to ataxia onset can reliably assess early executive dysfunctions, which cannot be evaluated with the saccadic slowing. Imaging Findings Regarding MRI imaging, the major findings were that the ponto-cerebellar system becomes atrophied some years before the disease onset in SCA2, confirming previous reports [14, 31] and that the cerebellar vermis atrophy was tightly associated to subtle motor impairments, which had not been demonstrated previously in non-ataxic carriers of any SCA mutations. Main experiences on the assessment and management of prodromal features for neurodegenerative diseases come from the Parkinson disease’s studies, which have identified multiple non-motor and/or slight motor features which occur before onset of typical motor symptoms in close association with specific anatomical patterns of Lewy body deposition in the Braak staging system [52, 53]. Thus, similarly to previous Parkinson diseases’ studies, in the present paper, we have recognized a group of early symptoms and signs that herald SCA2 various years before ataxia onset, which represent an important information for neurologists, neurophysiologists, and genetic counselors to assess the genetic liability to SCA2 mutation in at-risk descendants, improving their capabilities for diagnosis and to estimate the probable remaining time to ataxia onset. The understanding of prodromal SCA2 phenotype is also essential to design clinical trials with higher efficacy potential, because it offers the possibility to begin the therapies before ataxia onset and to identify sensitive biomarkers used as endpoint variables in these clinical trials. In conclusion, the recognition of prodromal SCA2 features offers novel insight into the physiopathological basis of SCA2 and subsequently improves the clinical management of the disease, broadening the diagnostic possibilities and promoting the design of clinical trials at early phases when therapeutical options should be most effective.

Acknowledgments We are grateful to the non-ataxic SCA2 mutation carriers and the control individuals, as well as to the Cuban Ministry of Public Health, the National Council of Science and Technology of Mexico (CONACyT), and the Ibero-Latin American network for Movement disorders (RIBERMOV) for their cooperation.

Conflict of Interest The authors declare that there are no conflicts of interest. Funding Statement This work was supported by the Cuban Ministry of Public Health and the CONACyT fellowship-203861 to L-VP. Contributorship Statement Luis Velázquez-Perez: Drafting the manuscript for content, study concept or design, acquisition of data, analysis or interpretation of data, study supervision or coordination, and final approval of the version to be published. Roberto Rodríguez-Labrada: Study concept or design, drafting the manuscript for content, acquisition of data, analysis or interpretation of data, study supervision or coordination, and final approval of the version to be published. Edilia M. Cruz Rivas: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Juan Fernandez-Ruiz: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Jandy Lilia-Campins: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Bulmaro Cisneros: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Israel Vaca-Palomares: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Arnoy Peña-Acosta: Acquisition of data and revising the manuscript for content. Yaimeé Vazquez-Mojena: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Rosalinda Diaz: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Jonathan J Magaña-Aguirre: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Tania Cruz-Mariño: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Annelié Estupiñan-Rodríguez: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. José M. Laffita-Mesa: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Rigoberto González-Piña: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Nalia Canales-Ochoa: Analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published. Yanetza Gonzalez-Zaldivar: Acquisition of data, analysis or interpretation of data, revising the manuscript for content, and final approval of the version to be published.

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