Caloric restriction blocks neuropathology and motor deficits in Machado–Joseph disease mouse models through SIRT1 pathway

Machado–Joseph disease (MJD) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the ATXN3 gene, translating into a polyglutamine tract within the ataxin-3 protein. The available treatments only ameliorate symptomatology and do not block disease progression. In this study we find that caloric restriction dramatically rescues the motor incoordination, imbalance and the associated neuropathology in transgenic MJD mice. We further show that caloric restriction rescues SIRT1 levels in transgenic MJD mice, whereas silencing SIRT1 is sufficient to prevent the beneficial effects on MJD pathology. In addition, the re-establishment of SIRT1 levels in MJD mouse model, through the gene delivery approach, significantly ameliorates neuropathology, reducing neuroinflammation and activating autophagy. Furthermore, the pharmacological activation of SIRT1 with resveratrol significantly reduces motor incoordination of MJD mice. The pharmacological SIRT1 activation could provide important benefits to treat MJD patients.


Supplementary
in the opposite side. The aquarium was filled up to the area of the platform with water at 24-26ºC.
The time that mice took to reach the platform was recorded. The time of four trials with an inter-trial interval of 60 seconds were recorded. The first trial was considered as an exploratory trial and the results were expressed as the average of the other three trials.
Beam walking test: In order to evaluate motor coordination and balance of mice, the ability of the mice to cross a graded series of narrow beams to reach an enclosed escape platform, was evaluated 3 . The test was performed in four long wood beams (1 m): two round beams with 9 or 6 mm diameter and two square beams with 18 or 9 mm square wide. The tests were performed sequentially from the widest to the narrowest beam: the first beam was the 18 mm square beam, followed by the 9 mm square beam and the 9 mm round beam, ending with the 6 mm round beam.
Each beam was placed horizontally, 25 cm above the bench surface, with one end attached on a narrow support and the other end fixed to a bounded box (20 cm square) into which the mouse could escape. Mice were allowed up to 60 sec to transverse each beam, and the time was recorded. Any animal that did not cross in 60 sec was assigned the maximum value of 60 sec for analysis.
Footprint pattern: Different parameters of gait can be evaluated simply and effectively tracking the footprint pattern. This test was used to evaluate the effect of caloric restriction in the gait of mice and was performed as we previously described 1 . To obtain footprints, mice feet were paint with black and white non-toxic paints, respectively in the hind and forefeet. Then, animals were allowed to walk along 100 cm long, in a 10 cm wide runaway fresh sheet of green paper, in an apparatus with 15 cm high walls. A fresh sheet of green paper was replaced on the floor for each new mouse. In order to analyze footprint patterns some parameters were explored (all measured in centimeters). Stride length, the average distance of forward movement between each stride, was measured. This parameter was determined by the measurement of the perpendicular distance of a given step to a line connecting its opposite preceding and proceeding steps. The average of three strides was obtained. To explore uniformity of step alternation the distance from left or right front footprint/hind footprint overlap was measured. A perfect overlap, recorded as zero, was considered when the centre of the hind footprint fell on the top of the centre of the preceding front footprint.
When the footprint did not overlap, the distance between the centre of footprints was verified. A sequence of four consecutive steps was chosen for evaluation, excluding footprints made at the beginning and the end of the run, where the animal was initiating or finishing the movement, respectively.
Open Field Test: Open-field test was used to explore locomotor horizontal activity and anxiety-like  (TABLE S1). Appropriate negative controls were also prepared. All reactions were performed in duplicate and according to the manufacturer's recommendations: 95ºC for 30 sec, followed by 45 cycles at 95ºC for 5 sec and 60ºC for 30 sec. The amplification rate for each target was evaluated from the cycle threshold (Ct) numbers obtained with cDNA dilutions, with correction for GADPH levels. The mRNA fold increase or fold decrease with respect to control samples was determined by the Pfäffl method.