Caffeine Alleviates Progressive Motor Deficits in ... - Wiley Online Library

RESEARCH ARTICLE

Caffeine Alleviates Progressive Motor Deficits in a Transgenic Mouse Model of Spinocerebellar Ataxia N elio Gonçalves, PhD,1,2 Ana T. Sim~ oes, PhD

,1,2 Rui D. Prediger, PhD,1,3

Hirokazu Hirai, MD, PhD,4 Rodrigo A. Cunha, PhD,1,5 and Luıs Pereira de Almeida, PhD

1,2

Objective: Machado–Joseph disease (MJD) is a neurodegenerative spinocerebellar ataxia (SCA) associated with an expanded polyglutamine tract within ataxin-3 for which there is currently no available therapy. We previously showed that caffeine, a nonselective adenosine receptor antagonist, delays the appearance of striatal damage resulting from expression of full-length mutant ataxin-3. Here we investigated the ability of caffeine to alleviate behavioral deficits and cerebellar neuropathology in transgenic mice with a severe ataxia resulting from expression of a truncated fragment of polyglutamine-expanded ataxin-3 in Purkinje cells. Methods: Control and transgenic c57Bl6 mice expressing in the mouse cerebella a truncated form of human ataxin-3 with 69 glutamine repeats were allowed to freely drink water or caffeinated water (1g/L). Treatments began at 7 weeks of age, when motor and ataxic phenotype emerges in MJD mice, and lasted up to 20 weeks. Mice were tested in a panel of locomotor behavioral paradigms, namely rotarod, beam balance and walking, pole, and water maze cued-platform version tests, and then sacrificed for cerebellar histology. Results: Caffeine consumption attenuated the progressive loss of general and fine-tuned motor function, balance, and grip strength, in parallel with preservation of cerebellar morphology through decreasing the loss of Purkinje neurons and the thinning of the molecular layer in different folia. Caffeine also rescued the putative striatal-dependent executive and cognitive deficiencies in MJD mice. Interpretation: Our findings provide the first in vivo demonstration that caffeine intake alleviates behavioral disabilities in a severely impaired animal model of SCA. ANN NEUROL 2017;81:407–418

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achado–Joseph disease (MJD), or spinocerebellar ataxia type 3, is a polyglutamine neurodegenerative disease and the most common among the group of dominantly inherited ataxias,1 a group of disorders also designated as spinocerebellar ataxias (SCAs). It results from an unstable expansion of a CAG stretch (over 55 repeats) in the ATXN3/MJD1 gene, which encodes a polyglutamine repeat in the ataxin-3 protein,1 and there is currently no available therapy. MJD has an adult age of onset and its clinical hallmarks include progressive ataxia, motor incoordination, postural instability, and Parkinsonism among other symptoms.2 The neuropathology involves multiple

systems, mainly cerebellar systems and also substantia nigra and cranial nerve motor nuclei,1,3 as well as the striatum.4,5 Several animal models closely mimicking the human pathology have been exploited to detail the mechanisms leading to neuronal degeneration,5–7 which have allowed us to highlight new targets for therapy such as impaired autophagy,8 proteolysis,9 and dysregulated peptide hormones10 preceded by synaptotoxicity and gliosis.11 One candidate target is adenosine A2A receptors (A2ARs), which afford protection in different neurodegenerative disorders.12,13 Accordingly, we recently

View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24867 Received May 1, 2016, and in revised form Aug 3, Nov 23, Nov 28, and Dec 12, 2016. Accepted for publication Dec 18, 2016. Address correspondence to Dr Pereira de Almeida, CNC–Center for Neuroscience and Cell Biology, University of Coimbra, 3004-504 Coimbra, Portugal. E-mail: [email protected] From the 1Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Faculty of Pharmacy, University of Coimbra, Coimbra, opolis, SC, Brazil; 4Department of Neurophysiology, Gunma Portugal; 3Department of Pharmacology, Federal University of Santa Catarina, Florian University Graduate School of Medicine, Maebashi, Gunma, Japan; and 5Faculty of Medicine, University of Coimbra, Coimbra, Portugal

C 2016 American Neurological Association V 407

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reported that caffeine consumption (an antagonist of adenosine receptors) attenuates striatal neuropathology in a MJD model.11 We here investigated whether chronic

caffeine consumption would alleviate the severe sensorimotor behavioral impairment in a transgenic mouse model7 expressing a truncated mutant ataxin-3 in

FIGURE 1.

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Purkinje cells, a model that can be considered a general SCA model, given the exhibited robust Purkinje cell degeneration.

Materials and Methods Animals and Treatments All experiments were approved by the Ethical Committee of the Center for Neuroscience and Cell Biology of Coimbra (CNC). C57Bl/6-background truncated-atx3-69Q–expressing transgenic MJD (TgMJD) mice, initially obtained from Gunma University,7 and wild-type (WT) littermates were bred at CNC. At 7 weeks of age, TgMJD (n 5 26) and WT (n 5 29) mice were randomly assigned to each experimental group and further provided with water (TgMJD, n 5 16; WT, n 5 16) or caffeine (TgMJD, n 5 10; WT, n 5 13) administered through the drinking water at a maximally effective and nontoxic dose (1g/L), which we have previously shown to result in a plasma concentration of 50 mM12 and similar concentration in the brain parenchyma.13 No animals were excluded either prior or after analysis unless due to technical issues regarding the video recording. Five TgMJD and 4 WT mice (chosen randomly) were killed at 7 weeks of age for histological and neurochemical processing; 4 mice of each experimental group (chosen randomly) were killed at 8 weeks of treatment to assess histological modifications; the remaining animals were killed after 20 weeks of treatment. The number of mice per group and the time points for analysis were based on our previous studies.10,14

Behavioral Assessments All tests were carried out between 9:00 and 17:00 hours in an observation sound-attenuated room under low-intensity light (12 lux), where mice had been habituated for at least 1 hour. Animals were tested by an operator unaware of genotype or treatment; behavior was monitored through a video camera positioned above the apparatuses, and the videos were later analyzed by an operator unaware of genotype or treatment. The apparatuses were cleaned with 10% ethanol between animals. Mice were repeatedly tested every 4 weeks, and the data in all

figures correspond to the same groups of mice, with exclusion of animals from analysis done due to poor video footage. General motor coordination was assessed as previously described,15 using an accelerating rotarod starting at 4rpm and accelerating to 40rpm over a period of 5 minutes to record the time each mouse remained in the rotation drum, calculated as the average of 2 trials with a 20-minute intertrial interval. Locomotion was assessed in an open field during 15 minutes,16 where maximum velocities were taken for analysis. Balance was evaluated in the pen test, where we recorded in a 60-second trial the ability of mice to grab for the pen (9mm diameter, horizontally fixed 25cm above the ground) and start walking on it.16 The mouse limb strength was measured using the grip test,17 where a mouse was hung with its forepaws to the central position of a 300g metal grid and its strength was determined as the weight pushed (in grams); the assay was performed 3 times, and the mean was considered for analysis. Subtle alterations of motor coordination and balance were assessed in the beam-walking test.17 Mice were allowed to traverse a graded series of narrow beams to reach an enclosed safety platform to escape an aversive stimulus (60W light) placed near the start of the beam. Mice were initially trained over 3 days (4 trials per day) to traverse the 9mm square beam before being tested in 2 consecutive trials on each of the square and round beams, progressing from the widest to the narrowest beam, with a maximum time per trial of 60 seconds. Any animal that did not cross within 60 seconds was allocated a maximum value of 60 seconds for analysis. Striatal-dependent motor coordination was further evaluated with the pole test,18 where a mouse was placed headupward on the top of a vertical rough-surfaced pole (diameter 5 1.0cm, height 5 52cm) and the time to orient downward (t-turn) and to reach the floor (t-descend) were recorded with a maximum allowed period of 120 seconds; mice were subjected to 5 consecutive trials with an intertrial interval of 60 seconds, and the best 3 scores for each parameter were considered for analysis. Striatal-dependent procedural learning was assessed using a cued version of the water maze,19 where mice were subjected

FIGURE 1: Chronic caffeine intake attenuates the impaired motor coordination in Machado–Joseph disease transgenic (TgMJD) mice. (A) On an accelerating rotarod, TgMJD mice (n 5 7–20) performed poorly (***p < 0.001, 2-way analysis of variance [ANOVA]) when compared to wild-type (WT) littermates (n 5 8–18) and worsened with age (p 5 0.06, n 5 7, Student t test); caffeine intake significantly attenuated this deficit in TgMJD mice (#p < 0.05, n 5 6, Student t test). (B) On a pen test, TgMJD mice displayed increasing balance impairment with age (*p < 0.05, n 5 5–7, Kruskal–Wallis test), which was significantly prevented by caffeine intake (#p < 0.05, n 5 5, Mann–Whitney test). (C-F) Latency of WT and TgMJD mice to cross a series of beams of square and round cross-section, progressing from the widest to the narrowest, to reach a safety platform. (C) TgMJD mice exhibited a decline in beam-walking ability with increasing beam difficulty and age (*p < 0.05, **p < 0.01, ***p < 0.001, n 5 5–8, Student t test with Welch correction). (D, E) TgMJD mice displayed a progressive beam-walking disability with age (*p < 0.05, **p < 0.01, ***p < 0.001 compared to TgMJD at 0 weeks of treatment, 1-way ANOVA), and 8 weeks but not 20 weeks of caffeine intake prevents (#p < 0.05, Student t test) the worsening of motor performance in the more challenging round beams. (F) At 27 weeks of age, TgMJD mice began displaying a subtle impairment (p 5 0.09, n 5 5–6, Student t test) in the narrowest square beam (arrow), which was not seen upon 20 weeks of caffeine administration. (G) In the pole test, older TgMJD mice showed, when compared to WT animals, significantly increased time to orient downward (**p < 0.01, n 5 7–8, Student t test with Welch correction), which was prevented by caffeine treatment (#p < 0.05, n 5 6, Student t test with Welch correction); TgMJD mice also displayed significantly increased time to descend to the floor (*p < 0.05, **p < 0.01; n 5 5–8, Student t test with Welch correction), an effect prevented by caffeine intake (#p < 0.05, n 5 6, Student t test), which restored mice to WT levels (&p < 0.01, Student t test relative to TgMJD, 0 weeks of treatment).

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to 4 training days, 4 consecutive trials per day, during which the mice learned to swim in a 140cm diameter pool toward a submerged platform tagged with a 5cm yellow rubber ball protruding above the water surface; the scores for latency to reach the platform were measured, and mice were gently guided to the platform when they took >60 seconds to reach it. Short-term spatial memory was assessed using the object displacement test,18 where we scored the time interacting with 2 identical objects (plastic columns with 4cm height and 5cm diameter) during 5 minutes in a session carried out 3 hours after a similar previous exposure to these 2 objects, where 1 of the objects had a different localization in the arena; a location index was calculated as follows: (Tnovel 3 100) / (Tnovel 1 Tfamiliar), where Tnovel is the time spent exploring the displaced object and Tfamiliar is the time spent exploring the nondisplaced object. Anxiety was evaluated in the elevated plus maze (four 40 3 5cm black acrylic cross-shaped arms raised 50cm above the floor), where anxiogenic-like effects were scored as decreased number of open arm entries over the total number of arm entries, and the time spent on open arms relative to the total time spent on both arms.18 Helpless behavior was assessed by scoring the immobility time of mice suspended by their tail during 6 minutes.18

Histological Assessments After an overdose of avertin (2.5 3 240 lg/g, intraperitoneal) and transcardial perfusion with 4% paraformaldehyde of half of the mice in each group (randomly chosen), the brains were coronally sectioned (25 lm thick) in anatomical series and freefloating sections were immunohistochemically processed as previously described10 to stain neurons with cresyl violet or Purkinje cells with calbindin (rabbit polyclonal; Chemicon, Temecula, CA) together with 4,6-diamidino-2-phenylindole (Sigma, St Louis, MO). After acquiring images with an Axiovert 200 microscope (Carl Zeiss, Oberkochen, Germany) equipped with AxioCam HR color digital cameras (Carl Zeiss) using 3 5, 3 20, and 340 Plan-Neofluar objectives, quantitative analysis was performed with a semiautomated image analysis software package (ImageJ software; NIH, Bethesda, MD).

A2AR Expression and Density Half of the mice per group (randomly chosen) were killed by cervical dislocation, and cerebella were dissected. Total mRNA was isolated using the kit NucleoSpin RNA (Macherey-Nagel, D€ uren, Germany), and quantitative polymerase chain reaction compared the A2AR gene (NM_009630) with reference genes (Hprt, NM_013556; Gapdh, NM_008084), as previously described.20 A2AR binding was carried out with 6nM of 3HSCH58261 in synaptosomal membranes, as previously described.12

Statistical Analysis Statistical comparisons were performed by unpaired parametric Student t test and 1-way or 2-way analysis of variance of multiple experimental groups followed by Dunnett multiple comparison or Bonferroni comparison post hoc tests, respectively, and

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TABLE 1. Analysis of the Neuromuscular Function of Machado-Joseph Disease transgenic mice Using a Grip Strength Test Weeks of Treatment

0

8

20

H2O Caffeine

72.0 6 3.1

48.5 6 9.1a 78.3 6 7.7b

47.9 6 4.6 44.2 6 6.0

Grip strength is expressed as weight pushed (in grams) from the scale (mean 6 standard error of the mean). a Difference from the initial value (p < 0.05; 1-way analysis of variance). b Difference from control aged-matched water-drinking group (p < 0.05; Student t test).

nonparametric Mann–Whitney and Kruskal–Wallis tests followed by Dunn multiple comparison post hoc test. Results are expressed as mean 6 standard error of the mean or median and interquartile range (IQR), and significance thresholds were set at p < 0.05, p < 0.01, and p < 0.001, as defined in the text.

Results Caffeine Prevents the Impairment of Motor Coordination and Balance TgMJD mice express a truncated form of human ataxin3 with 69 glutamine repeats in the mouse cerebella, causing a severe ataxic phenotype associated with cerebellar defects.7 Accordingly, in the rotarod test, which is especially sensitive to quantify the progressive sensorimotor impairments associated with cerebellar dysfunction, TgMJD mice displayed a severe deficit, which was present as early as 7 weeks of age (p < 0.001) and progressively deteriorated with age, whereas WT mice displayed a stable performance (Fig 1A). Chronic caffeine consumption attenuated (p < 0.05) the progressive loss of TgMJD mouse performance on the rotating rod, whereas it was devoid of effects in WT mice (see Fig 1A). Because the rotarod paradigm depends on muscle strength, coordination, and balance, we detailed the nature of the rotarod impairment. In the pen test, TgMJD mice displayed increasing difficulties in maintaining their equilibrium over the beam (see Fig 1B), a phenotype that was aggravated in older mice (p < 0.05). Notably, caffeine prevented these balance deficits (p < 0.05), to such an extent that the caffeine-drinking TgMJD mice performed as well as WT animals (median 5 60 seconds, IQR 5 8.25, n 5 8). Endurance was evaluated using a grip strength test. At 7 weeks of age (Time 0 in Table 1, n 5 4–7), TgMJD mice had muscle strength similar to their WT littermates (74.6 6 3.8g, n 5 6), but TgMJD mice developed a loss Volume 81, No. 3


TABLE 2. Analysis of TgMJD Locomotor Performance Using an Open-Field Apparatus Maximum Velocity, cm/s Mice

Time, wk 0

8

20

WT

H2O Caffeine

14.5 6 0.4 14.7 6 0.8

12.7 6 0.9 13.8 6 0.7

TgMJD H2O Caffeine

15.9 6 0.5

13.0 6 0.5a 12.7 6 0.5b 11.2 6 0.9 14.9 6 0.8c 11.9 6 2.3

Analysis of the maximum reached velocity (in centimeters per second) during 15-minutes test in the open field; data are expressed as mean 6 standard error of the mean. Statistical evaluation of the genotype was made by 2-way analysis of variance (ap < 0.001, b p < 0.05). Caffeine effects were evaluated by Student t test (cp < 0.05). TgMJD 5 Machado–Joseph Disease transgenic mice; WT 5wild type.

of grip strength (p < 0.05) within the subsequent 8 weeks, which was prevented by caffeine intake (p < 0.05). However, the prolonged caffeine treatment was unable to sustain the initial endurance of TgMJD mice up to 20 weeks (see Table 1). Interestingly, TgMJD mice also displayed a decrease (p < 0.001) of maximum velocities when spontaneously exploring an open field apparatus (Time 0 in Table 2, n 5 9–13), an effect also prevented (p < 0.05) by 8 weeks of caffeine treatment. Caffeine Improves Fine Motor Function Whereas the rotarod is useful for determining gross motor deficits in rodents, the detection of subtler motor effects requires other tests such as the beam-walking task, which examines the ability of the animal to remain upright and to walk without falling on 4 square and round cross-section elevated beams with progressively smaller diameter, to reach a safety platform.21 At 7 weeks of age, TgMJD mice already displayed an impaired performance (p < 0.05) on the narrowest square and round beams, which significantly worsened with age in both the narrowest and wider round (p < 0.001) and square (p < 0.01) beams (Fig 1C). At 15 weeks of age, waterdrinking TgMJD mice hardly performed the task on rounded beams, and older mice (20 weeks, waterdrinking group) began exhibiting an additional subtle impairment (p 5 0.09) on the narrowest square beam (see Figs 1D,E,F). Caffeine intake prevented the evolving inability of 15-week-old TgMJD mice (8 weeks of caffeine drinking) to perform the widest (p < 0.05) and the narrowest (p < 0.05) rounded beams. This alleviation of the phenotype observed in rounded beams was no longer March 2017

observed at 27 weeks of age (20 weeks of treatment). Nevertheless, caffeine-treated TgMJD mice did not show worsening of performance on the narrowest square beam at this later age. Caffeine Prevents the Impairment of StriatalDependent Motor Performance To probe whether, apart from the expected cerebellar dysfunction, the motor coordination deficits displayed by TgMJD mice also involved an altered nigrostriatal circuitry, we subjected the animals to the pole test. The time required by older TgMJD mice to orient downward was longer (p < 0.01) than their WT littermates (see Fig 1G); the time taken by TgMJD mice to descend the pole was also longer than WT mice at both younger (p < 0.01) and older ages (p < 0.05). As shown in Figure 1G, caffeine-treated TgMJD mice descended the pole faster (p < 0.05) and actually recovered (p < 0.01) to the performance of WT mice (4.7 6 0.7 seconds vs 4.2 6 0.5 seconds, p 5 0.56, n 5 6–8); caffeine intake also completely prevented the increased time of TgMJD mice to orient downward (p < 0.05). We next gauged the presence in TgMJD mice of procedural learning and memory deficits, because we noted a mild learning impairment at day 2 of training in the raised narrow beam task in 15-week-old TgMJD mice (8 weeks water-drinking group), which was not seen in caffeine-treated animals (Fig 2). We used a water maze cued-platform acquisition test to score the escape latency within trials as a measure of procedural learning. We only found differences in TgMJD mice at 13 weeks of age (6 weeks of water drinking), which took longer than WT mice (p < 0.01) to reach the safety platform at day 1. Again, caffeine prevented (p < 0.01) the increased time taken by TgMJD mice to find the platform at day 1, as was observed at day 2 in the beam-walking task. Presumably because the survival instinct induced a rapid search for the safety platform, all animals performed equally well despite the motor incoordination of TgMJD mice, which impeded determining whether TgMJD mice displayed a putative impairment of this striataldependent form of learning. Thus, we used the object displacement test to probe procedural spatial memory impairments, exploring the spontaneous tendency of rodents to explore novel stimuli, here a displaced object. During the training phase, all mice spent an equal amount of time exploring each of the 2 identical objects (from 55.8 6 5.3 seconds to 76.4 6 10.8 seconds, p > 0.05, n 5 6–9). As expected, WT mice displayed a location index different (p < 0.05) from the chance level, whereas TgMJD mice had a reduced location index, not different from the chance level. Moreover, WT mice 411

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FIGURE 2: Chronic caffeine intake attenuates the impaired motor learning and executive procedural memory performance of Machado–Joseph Disease transgenic (TgMJD) mice, which did not display an impaired mood-related performance. (A) The acquisition of motor coordination learning in the beam-walking test showed that 15-week-old TgMJD mice display a learning impairment at day 2 when performing the narrowest 9mm square beam, which did not occur either in caffeine-treated TgMJD animals or in wild-type (WT) mice, whose excellent performance precluded this observation. (B) In a swimming task (cued-version of the water maze) using a similar training protocol, 13-week-old TgMJD mice already displayed a significant initial akinesia compared to WT (**p < 0.01, n 5 4–8, 2-way analysis of variance [ANOVA]), which was prevented by caffeine treatment (##p < 0.01, n 5 3, 2-way ANOVA). (C) In the object displacement test, WT mice spent more time exploring the displaced object than the object placed in the same position as in the acquisition trial (*p < 0.05, n 5 8, 1-sample t test, chance level of 50%), whereas TgMJD mice poorly discriminate the displaced object (p > 0.05, n 5 7; ie, not different from the chance level). Caffeine intake prevented the inability of aged TgMJD mice to explore novelty (#p < 0.05, n 5 6 vs TgMJD mice drinking water) and did not change the WT performance (**p < 0.01, n 5 9, 1-sample t test). (D, E) In the elevated plus maze, TgMJD mice displayed a significantly (*p < 0.05, n 5 7–8, Student t test) greater percentage of time (D) and a greater number of entries (E) in the open arms than WT mice, suggestive of anxiolytic behavior in TgMJD compared to WT mice. (F) A significant reduction (***p < 0.001, n 5 7–8, Student t test) in the immobility time of TgMJD mice compared to WT mice was observed in the tail suspension test, suggestive of decreased helpless behavior.

decreased their exploration time in the test trial (44.3 6 8.7 seconds) compared to the training phase (62.5 6 8.1 seconds, p 5 0.07, n 5 8), consistent with their ability to recognize the environment, whereas TgMJD mice spent the same time exploring the objects in the 2 trials (76.4 6 10.8 seconds and 74.1 6 22.6 seconds, p 5 0.93, n 5 7). Importantly, caffeine treatment rescued (p < 0.05) the inability of aged TgMJD mice to 412

learn and therefore to explore novelty, whereas it did not change the performance of WT mice (p < 0.01 vs chance level). TgMJD Mice Do Not Display Mood-Related Impairments We carried out a preliminary survey of alterations of mood-related behavior in TgMJD mice by assessing Volume 81, No. 3


FIGURE 3: Machado–Joseph Disease transgenic (TgMJD) mice display cerebellar atrophy, which is attenuated by chronic caffeine intake. (A) Representative brains from wild-type (WT) and TgMJD littermate mice; scale bar 5 1mm. (B) Quantification of the extrapolated hemicerebellar volume of young TgMJD mice showing a marked reduction of the cerebellar size (***p < 0.001, n 5 4, Student t test) when compared to aged-matched WT mice, a reduction even more pronounced in the older water-drinking TgMJD mice, which was prevented by 8 weeks of caffeine treatment (#p < 0.05, n 5 4, Student t test). (C) Midsagittal cresyl violet–stained sections from 15-week-old TgMJD mice drinking water (top) or caffeinated water (bottom). Arrows indicate foliation defects: decreased size of V, VIII, and IX. Scale bar 5 200 lm. [Color figure can be viewed at wileyonlinelibrary.com]

anxiety-related behavior in the elevated plus maze and helpless behavior in the tail suspension test.18 At 27 weeks of age, TgMJD mice displayed anxiolytic behavior, typified by an increased percentage of time and increased number of entries in the open arms of an elevated plus maze compared to aged-matched WT littermates (p < 0.05; see Fig 2). TgMJD mice also presented decreased helpless behavior (p < 0.001), as concluded by the decreased immobility time in the tail suspension test compared to age-matched WT littermates. Caffeine Rescues the Progression of Cerebellar Neuropathology We next directly investigated whether caffeine decreased cerebellar neuropathology in TgMJD mice. Consistent with the previously reported gross morphological defects in Purkinje cells,7 TgMJD mice displayed reduced cerebellar size (Fig 3). Cresyl violet staining confirmed that 7-weekold TgMJD mice showed reduced cerebellar size (p < 0.001) when compared to WT mice, which increased even more with aging. This reduction was associated with an indistinguishable neocerebellum folia (VI and VII), already observed at 7 weeks of age, in parallel with an agedependent reduction of paleocerebellum folia V, VIII, and IX. Importantly, chronic caffeine intake rescued (p < 0.05) this cerebellar global shrinkage in TgMJD mice. March 2017

When investigating the different molecular layers, we found that TgMJD mouse cerebella exhibited a significant (p < 0.001) decrease in the thickness of the molecular layers in different folia (lobule V: 68 6 6 lm; lobule IX: 67 6 5 lm) compared to their WT littermates (lobule V: 137 6 4 lm; lobule IX: 155 6 3 lm) as early as 7 weeks of age (Fig 4). At 27 weeks of age, the cerebellar molecular layers of water-drinking TgMJD mice thinned to 52 6 3 lm (lobule V, p < 0.05) and 48 6 2 lm (lobule IX, p < 0.01), which was accompanied by a disorganization of the Purkinje cell layer. The chronic intake of caffeine (up to 20 weeks) afforded a preservation (p < 0.05) of the molecular layer thickness in TgMJD mice. To ascertain whether this thinning of the cerebellar molecular layers involved a putative loss of Purkinje cells, we quantified the number of immunofluorescent calbindin-positive cells, a marker of Purkinje neurons (see Fig 4C, arrows). Young adult TgMJD mice (7 weeks of age) displayed a lower number of Purkinje neurons in both lobules V (p < 0.001) and IX (p < 0.05) when compared to WT mice (see Fig 4D), which progressively worsened with age (lobule V: p < 0.01; lobule IX: p 5 0.10). Notably, caffeine intake attenuated the loss of calbindin-immunofluorescent neurons in both lobules V (p < 0.01) and IX (p < 0.05) in TgMJD mice. 413

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Because caffeine exerts its neuroprotective effects through the antagonism of neuronal A2ARs that are upregulated in brain diseases22,23 and TgMJD mice display neuronal loss in the cerebellum, we tested whether A2AR density was altered in the cerebellum of TgMJD mice. As shown in Figure 5A, there was a 33% (p 5 0.16) increased expression of A2AR mRNA in the cerebellum of TgMJD mice at 7 weeks of age compared to WT animals, and increased density (p < 0.001) of the A2AR protein in synaptic membranes (see Fig 5B), as assessed by the binding density of the selective A2AR antagonist 3HSCH58261 (6nM) in cerebellar synaptosomal membranes from TgMJD compared to WT mice.

Discussion The present study shows that chronic caffeine intake delayed the onset of the motor deterioration that worsens with time in a transgenic mouse (TgMJD) expressing a human form of ataxin-3 with 69 CAG repeats in the cerebellum.7 This is paralleled by an ability of caffeine to rescue the progression of morphological damage and neurodegeneration in the cerebellum of TgMJD mice. This combined behavioral and morphological evidence heralding the neuroprotective capacity of caffeine in MJD prompts two novel working hypotheses pertinent to the neurobiological basis and the management of MJD: on one hand, it points to a key role of the target for caffeine neuroprotection, namely A2ARs, in the evolution of cerebellar neurodegeneration in MJD, as has been shown in other brain regions in the realm of other neurodegenerative disorders22–24; on the other hand, it suggests a simple and testable lifestyle measure to alleviate MJD symptoms, that is, the consumption of caffeinated beverages, as has been proposed for other brain conditions.22–24 The morphological evaluation of cerebellar damage in TgMJD showed an evident and rapidly evolving atrophy of the cerebellum that involved a marked neuronal loss, namely of the output neurons of the cerebellum

(ie, Purkinje cells). Notably, chronic caffeine consumption markedly attenuated this cerebellar damage putatively due to the blockade of A2ARs, because A2ARs are the molecular target of chronic caffeine used to efficiently prevent neurodegeneration in the hippocampus of animal models of Alzheimer disease25 or early life convulsions,26 as well as striatal degeneration upon intrastriatal injection of mutant ataxin-3.11 Furthermore, it was previously shown that caffeine prevented the damage of cerebellar neurons in a manner mimicked by selective A2AR antagonism.27,28 Given that A2AR activation is sufficient to trigger brain dysfunction,29 the presently observed upregulation of A2ARs in cerebellar synapses of TgMJD mice reinforces our previous proposal that synaptotoxicity might be an early feature in MJD,11 as shown to occur in other neurodegenerative disorders,30,31 which can be counteracted by caffeine to dampen the deterioration of brain function.22–24 This cerebellar degeneration and its control by caffeine were of significant phenotypic relevance because the cerebellar-dependent phenotypic changes characteristic of MJD were also attenuated by the chronic consumption of caffeine. At early stages of cerebellar degenerative disorders, the motor dysfunction typified by impaired gait (ataxia) and extremity incoordination (dysmetria) is preceded by poor balance and inability to walk in a straight line, and is commonly accompanied by impaired force of contraction.32 Accordingly, TgMJD mice displayed a marked and progressive sensorimotor impairment in the rotarod and pen tests correlated with an early loss of muscle strength. Walking also requires fine motor coordination, and we used the beam-walking test to assess walking abilities in graded levels of difficulty.21 TgMJD mice showed greater difficulty in performing the beamwalking test when challenged on the narrowest rounded beams, compared to the widened squared beams. Chronic caffeine consumption attenuated this early loss of performance on both mildly difficult (rotarod and pen test) and highly difficult tasks (beam walking), indicating that

FIGURE 4: Chronic caffeine intake attenuates the atrophy of cerebellar layers and the degeneration of Purkinje neurons in Machado–Joseph Disease transgenic (TgMJD) mice. (A) Cresyl violet–stained midsagittal sections of both TgMJD and wild-type (WT) littermate mice; scale bar 5 50 lm. GL 5 Granular layer; ML 5 Molecular layer; PCL 5 Purkinje cell layer. (B) Quantification of the thickness of the ML of V and IX folia, showing that 7-week-old TgMJD mice (n 5 4) displayed a significantly thicker ML (***p < 0.001, Student t test) when compared to WT mice (n 5 4), irrespective of the folia; upon aging, water-drinking TgMJD mice showed a progressive reduction of ML thickness (lobule V, *p < 0.05; lobule IX, **p < 0.01; n 5 5, Student t test; vs 7week-old TgMJD mice) together with an increased disorganization of the PCL (A). Notably, 20 weeks of caffeine treatment significantly slowed (#p < 0.05, n 5 5, Student t test) the progressive reduction of the ML thickness in TgMJD mice. (C) The immunostaining of cerebellar Purkinje neurons with an anticalbindin antibody showed a significant reduction of the number of immunoreactive Purkinje neurons in 7-week-old TgMJD mice when compared to aged-matched WT littermates; scale bar 5 50 lm. (D) This is quantified in both the anterior (lobule V, ***p < 0.001) and posterior (lobule IX, *p < 0.05; n 5 4, Student t test) lobes. It was even more reduced in aged water-drinking TgMJD animals (lobule V, **p < 0.01; lobule IX, p 5 0.10; n 5 5, Student t test); caffeine intake during 20 weeks completely abolished the loss of Purkinje neurons in both lobes (lobule V, ##p < 0.01; lobule IX, #p < 0.05; n 5 5, Student t test). [Color figure can be viewed at wileyonlinelibrary.com]

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FIGURE 5: Upregulation of adenosine A2A receptors (A2AR) in cerebellar nerve terminals of Machado–Joseph Disease transgenic (TgMJD) mice. (A) Increased levels of A2AR mRNA relative to Gapdh in extracts from the cerebellum of 7-week-old TgMJD compared to wild-type (WT) littermate mice (1.33 6 0.14 vs 1.00 6 0.13, p 5 0.16, Student t test, n 5 3–4). (B) Increased density (***p < 0.001, Student t test) of the number of binding sites of the selective A2AR antagonist 3H-SCH58261 (6nM) to synaptosomal membranes of 7week-old TgMJD (51.3 6 1.8fmol/mg protein, n 5 5) compared to WT littermate mice (22.6 6 2.1fmol/mg protein, n 5 4). Data are mean 6 standard error of the mean.

caffeine prevents the loss of motor coordination of TgMJD mice when executing more challenging and physically demanding motor coordination tests. This conclusion is in agreement with the previously reported ability of the chronic (not acute) intake of caffeine to dampen motor incoordination associated with alcohol consumption33 and to decrease developmental coordination disorder in children suffering from apnea of prematurity.34 This prompts a renewed interest in exploring the role of the scarcely abundant A2ARs in the cerebellum35,36 in the control of information flow in cerebellar circuits. Despite the cerebellum being traditionally viewed as a motor coordination center, it is also linked to nonmotor regions of the cerebral cortex and plays a role in executive functions such as cognitive planning.37 Clinical neuropsychological tests reveal that confined cerebellar degeneration is associated with deficits in strategy formation, procedural learning, the memory for motor skills, and stimulus-response habits.37,38 Accordingly, MJD animal models and patients39,40 exhibit hallmarks of cerebellar cognitive affective syndrome involving impaired executive and attentional features,40 as well as cognitive dysfunction.41 Thus, we explored whether more integrative behaviors involving cerebellar function that are characteristically affected in MJD were also modified in TgMJD mice and whether they were ameliorated by caffeine. We observed that TgMJD mice displayed deficits of procedural learning and executive memory, as revealed by their poorer performance in the cued version of the water maze and in the object displacement tests. 416

Importantly, these deficits were normalized by chronic caffeine intake, further heralding a general benefit resulting from regular caffeine intake to alleviate the burden of MJD. These effects of caffeine are likely associated with the A2AR-mediated neuroprotection resulting from longterm caffeine intake, rather than resulting from the A1Rmediated stimulant or attentional effects of acute caffeine administration,42,43 which undergo rapid tachyphylaxis.44 Notably, the analysis of TgMJD brains showed a decreased neocerebellum (vermis and lobules VI and VII), which is responsible for higher level cognitive/emotional tasks such as planning, initiation and timing of movements, and reduced anterior–posterior lobules (V and VIII to X), which are associated with sensorimotor functions connected to the cerebral cortex and spinal cord, and, thus, related to fine-tuning body and limb movements as well as muscle tone, balance, and postural stability. Therefore, the benefits afforded by chronic caffeine intake could result from its ability to attenuate the cerebellar neuropathology of TgMJD mice. However, the exploration of novel environmental stimuli also depends on the integrity of limbic and nonlimbic pathways, including the basal forebrain, hippocampus, thalamus, prefrontal cortex, and dorsal striatum, as well as the vestibular system and cerebellum.45 This prompts the alternative hypothesis that TgMJD mice might display a generalized impairment of different brain circuits, most of which can be normalized by chronic caffeine intake, in accordance with the localization of A2ARs in different brain areas.35,36 However, we observed that TgMJD mice displayed anxiolytic and lower helpless behavior, in contrast to MJD patients,46 which suggests that the deterioration of brain circuits might be limited to motor and executive functions rather than being generalized. Taken together, these present observations provide the first direct demonstration of the ability of chronic caffeine consumption to prevent the deterioration of a severe ataxic condition by rescuing the cerebellum from neurodegeneration and restoring the normal operation of the brain of MJD mice. However, this conclusion was based on the exploration of a particular MJD mouse model with selective overexpression of mutant ataxin-3 in the cerebellum, in contrast to the more widespread expression in MJD patients. In this model, the expression of truncated mutant ataxin-3 is controlled by the L7 promoter, specific for Purkinje cells. Despite evidence of variable Purkinje cell degeneration in MJD patients and models,47,48 this could lead to exacerbation of the disease in these cells compared with what is observed in human neuropathology, and therefore the observations may actually be even more relevant in the context of pure SCAs, Volume 81, No. 3


in which the neurodegeneration occurs primarily in Purkinje cells. It is also important to note that caffeine was more effective in affording benefits at early rather than at later time points analyzed; this indicates a main ability of caffeine to delay rather to abrogate MJD-related behavioral deficits. It remains to be determined what might be the long-term consequences of caffeine intake, that is, whether the purported caffeine-induced neuroprotection persists over time. This could be tackled in epidemiological studies, so far unavailable, exploring the impact of caffeine intake on the progression of MJD symptoms.

structures and basal ganglia in spinocerebellar ataxia types 1, 2 and 3. Brain 1998;121:1687–1693. 5.

Alves S, Regulier E, Nascimento-Ferreira I, et al. Striatal and nigral pathology in a lentiviral rat model of Machado-Joseph disease. Hum Mol Genet 2008;17:2071–2083.

6.

Cemal CK, Carroll CJ, Lawrence L, et al. YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit. Hum Mol Genet 2002;11:1075–1094.

7.

Torashima T, Koyama C, Iizuka A, et al. Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia. EMBO Rep 2008;9:393–399.

8.

Nascimento-Ferreira I, Santos-Ferreira T, Sousa-Ferreira L, et al. Overexpression of the autophagic beclin-1 protein clears mutant ataxin-3 and alleviates Machado-Joseph disease. Brain 2011;134:1400–1415.

9.

Sim~ oes AT, Gonçalves N, Koeppen A, et al. Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin-3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease. Brain 2012;35:2428–2439.

10.

Neves J, Gonçalves N, Cunha-Santos J, et al. Neuropeptide Y mitigates neuropathology and motor deficits in mouse models of Machado-Joseph disease. Hum Mol Genet 2015;24:5451–5463.

11.

Gonçalves N, Sim~ oes AT, Cunha RA, de Almeida LP. Caffeine and adenosine A2A receptor inactivation decrease striatal neuropathology in a lentiviral-based model of Machado-Joseph disease. Ann Neurol 2013;73:655–666.

12.

Duarte JM, Agostinho PM, Carvalho RA, Cunha RA. Caffeine consumption prevents diabetes-induced memory impairment and synaptotoxicity in the hippocampus of NONcZNO10/LTJ mice. PLoS One 2012;7:e21899.

13.

Costenla AR, Cunha RA, de Mendonça A. Caffeine, adenosine receptors, and synaptic plasticity. J Alzheimers Dis 2010;20(suppl 1):S25–S34.

14.

Nascimento-Ferreira I, N obrega C, Vasconcelos-Ferreira A, et al. Beclin 1 mitigates motor and neuropathological deficits in genetic mouse models of Machado-Joseph disease. Brain 2013;136:2173– 2188.

15.

N obrega C, Nascimento-Ferreira I, Onofre I, et al. Overexpression of mutant ataxin-3 in mouse cerebellum induces ataxia and cerebellar neuropathology. Cerebellum 2013;12:441–455.

16.

Boy J, Schmidt T, Wolburg H, et al. Reversibility of symptoms in a conditional mouse model of spinocerebellar ataxia type 3. Hum Mol Genet 2009;18:4282–4295.

17.

Sim~ oes AT, Gonçalves N, Nobre RJ, et al. Calpain inhibition reduces ataxin-3 cleavage alleviating neuropathology and motor impairments in mouse models of Machado-Joseph disease. Hum Mol Genet 2014;23:4932–4944.

18.

R.A.C. is a scientific consultant for the Institute for Scientific Information on Coffee.

Rial D, Castro AA, Machado N, et al. Behavioral phenotyping of Parkin-deficient mice: looking for early preclinical features of Parkinson’s disease. PLoS One 2014;9:e114216.

19.

Prediger RD, Aguiar AS Jr, Rojas-Mayorquin AE, et al. Single intranasal administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in C57BL/6 mice models early preclinical phase of Parkinson’s disease. Neurotox Res 2010;17:114–129.

References

20.

Sim~ oes AP, Machado NJ, Gonçalves N, et al. Adenosine A2A receptors in the amygdala control synaptic plasticity and contextual fear memory. Neuropsychopharmacology 2016;41:2862–2871.

21.

Carter RJ, Lione LA, Humby T, et al. Characterization of progressive motor deficits in mice transgenic for the human Huntington’s disease mutation. J Neurosci 1999;19:3248–3257.

22.

Chen JF, Eltzschig HK, Fredholm BB. Adenosine receptors as drug targets—what are the challenges? Nat Rev Drug Discov 2013;12:265–286.

23.

Cunha RA. How does adenosine control neuronal dysfunction and neurodegeneration? J Neurochem 2016;139:1019–1055.

Acknowledgment Supported by the European Regional Development Fund (ERDF) through the Operational Program Factors of Competitiveness (COMPETE, QREN grant CENTRO07-ST24-FEDER-002006), by national funds through the Portuguese Foundation for Science and Technology (FCT) grants SAU-FCF/70384/2006, E-Rare4/0003/2012, and SAU-NEU/099307/2008, strategic project UID/NEU/ 04539/2013, and fellowships SFRH/BD/38636/2007 (N.G.) and SRFH/BPD/87341/2012 (A.T.S.); Association Française Contre les Myopaties (AFM); National Ataxia Foundation; Santa Casa da Misericordia, Portugal; Brain & Behavior Research Foundation; the American-Portuguese Biomedical Research Fund and the Richard Chin and Lily Lock Machado-Joseph Research Fund; CAPESFCT; and CNPq (Science Without Borders).

Author Contributions R.A.C. and L.P.d.A. contributed to conception and design of the study. All authors contributed to acquisition and analysis of data. N.G., L.P.d.A., and R.A.C. contributed to drafting the manuscript and preparation of the figures.

Potential Conflicts of Interest

1.

Durr A, Stevanin G, Cancel G, et al. Spinocerebellar ataxia 3 and Machado-Joseph disease: clinical, molecular, and neuropathological features. Ann Neurol 1996;39:490–499.

2.

Taroni F, DiDonato S. Pathways to motor incoordination: the inherited ataxias. Nat Rev Neurosci 2004;5:641–655.

3.

Sudarsky L, Coutinho P. Machado-Joseph disease. Clin Neurosci 1995;3:17–22.

4.

Klockgether T, Skalej M, Wedekind D, et al. Autosomal dominant cerebellar ataxia type I. MRI-based volumetry of posterior fossa

March 2017

417

ANNALS

of Neurology

24.

de Mendonça A, Cunha RA. Therapeutic opportunities for caffeine in Alzheimer’s disease and other neurodegenerative disorders. J Alzheimers Dis 2010;20(suppl 1):S1–S2.

36.

Rosin DL, Robeva A, Woodard RL, et al. Immunohistochemical localization of adenosine A2A receptors in the rat central nervous system. J Comp Neurol 1988;401:163–186.

25.

Dall’Igna OP, Fett P, Gomes MW, et al. Caffeine and adenosine A2A receptor antagonists prevent b-amyloid (25-35)induced cognitive deficits in mice. Exp Neurol 2007;203:241– 245.

37.

Stoodley CJ, Schmahmann JD. Evidence for topographic organization in the cerebellum of motor control versus cognitive and affective processing. Cortex 2010;46:831–844.

26.

Cognato GP, Agostinho PM, Hockemeyer J, et al. Caffeine and an adenosine A2A receptor antagonist prevent memory impairment and synaptotoxicity in adult rats triggered by a convulsive episode in early life. J Neurochem 2010;112:453–462.

38.

Pascual-Leone A, Grafman J, Clark K, et al. Procedural learning in Parkinson’s disease and cerebellar degeneration. Ann Neurol 1993;34:594–602.

39.

27.

Dall’Igna OP, Porci uncula LO, Souza DO, et al. Neuroprotection by caffeine and adenosine A2A receptor blockade of beta-amyloid neurotoxicity. Br J Pharmacol 2003;138:1207–1209.

Zawacki TM, Grace J, Friedman JH, Sudarsky L. Executive and emotional dysfunction in Machado-Joseph disease. Mov Disord 2002;17:1004–1010.

40.

28.

Boeck CR, Kroth EH, Bronzatto MJ, Vendite D. Effect of the L- or D-aspartate on ecto-5’-nucleotidase activity and on cellular viability in cultured neurons: participation of the adenosine A2A receptors. Amino Acids 2007;33:439–444.

Maruff P, Tyler P, Burt T, et al. Cognitive deficits in MachadoJoseph disease. Ann Neurol 1996;40:421–427.

41.

Schmahmann JD, Sherman JC. The cerebellar cognitive affective syndrome. Brain 1998;121:561–579.

42.

Ferr e S. An update on the mechanisms of the psychostimulant effects of caffeine. J Neurochem 2008;105:1067–1079.

43.

Cunha RA, Agostinho PM. Chronic caffeine consumption prevents memory disturbance in different animal models of memory decline. J Alzheimer Dis 2010;20(suppl 1):S95–S116.

44.

Holtzman SG, Finn IB. Tolerance to behavioral effects of caffeine in rats. Pharmacol Biochem Behav 1988;29:411–418.

29.

Li P, Rial D, Canas PM, et al. Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory. Mol Psychiatry 2015;20:1339–1349.

30.

Conforti L, Adalbert R, Coleman MP. Neuronal death: where does the end begin? Trends Neurosci 2007;30:159–166.

31.

Milnerwood AJ, Raymond LA. Early synaptic pathophysiology in neurodegeneration: insights from Huntington’s disease. Trends Neurosci 2010;33:513–523.

45.

32.

Schmahmann JD. Disorders of the cerebellum: ataxia, dysmetria of thought, and the cerebellar cognitive affective syndrome. J Neuropsychiatry Clin Neurosci 2004;16:367–378.

Lalonde R. The neurobiological basis of spontaneous alternation. Neurosci Biobehav Rev 2002;26:91–104.

46.

33.

Fritz BM, Companion M, Boehm SL. “Wired,” yet intoxicated: modeling binge caffeine and alcohol co-consumption in the mouse. Alcohol Clin Exp Res 2014;38:2269–2278.

Braga-Neto P, Pedroso JL, Alessi H, et al. Cerebellar cognitive affective syndrome in Machado Joseph disease: core clinical features. Cerebellum 2012;11:549–556.

47.

34.

Doyle LW, Schmidt B, Anderson PJ, et al. Reduction in developmental coordination disorder with neonatal caffeine therapy. J Pediatr 2014;165:356–359.

Kumada S, Hayashi M, Mizuguchi M, et al. Cerebellar degeneration in hereditary dentatorubral-pallidoluysian atrophy and Machado-Joseph disease. Acta Neuropathol 2000;99:48–54.

48.

35.

Johansson B, Fredholm BB. Further characterization of the binding of the adenosine receptor agonist [3H]CGS 21680 to rat brain using autoradiography. Neuropharmacology 1995;34:393–403.

Switonski PM, Szlachcic WJ, Krzyzosiak WJ, et al. A new humanized ataxin-3 knock-in mouse model combines the genetic features, pathogenesis of neurons and glia and late disease onset of SCA3/MJD. Neurobiol Dis 2015;73:174–188.

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