SIRT1 overexpression ameliorates a mouse model of SOD1-linked amyotrophic lateral sclerosis via HSF1/HSP70i chaperone system

Background Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system. Results We established a line of transgenic mice that chronically overexpress SIRT1 in the brain and spinal cord. While inducible HSP70 (HSP70i) was upregulated in the spinal cord of SIRT1 transgenic mice (PrP-Sirt1), no neurological and behavioral alterations were detected. To test hypothetical benefits of SIRT1 overexpression, we crossbred PrP-Sirt1 mice with two lines of ALS model mice: A high expression line that exhibits a severe phenotype (SOD1G93A-H) or a low expression line with a milder phenotype (SOD1G93A-L). The Sirt1 transgene conferred longer lifespan without altering the time of symptomatic onset in SOD1G93A-L. Biochemical analysis of the spinal cord revealed that SIRT1 induced HSP70i expression through deacetylation of HSF1 and that SOD1G93A-L/PrP-Sirt1 double transgenic mice contained less insoluble SOD1 than SOD1G93A-L mice. Parallel experiments showed that Sirt1 transgene could not rescue a more severe phenotype of SOD1G93A-H transgenic mice partly because their HSP70i level had peaked out. Conclusions The genetic supplementation of SIRT1 can ameliorate a mutant SOD1-linked ALS mouse model partly through the activation of the HSF1/HSP70i chaperone system. Future studies shall include testing potential benefits of pharmacological enhancement of the deacetylation activity of SIRT1 after the onset of the symptom.

A mouse restrained in a cylinder was placed in the chamber of a startle reflex measurement system (O'Hara & Co.) with 70 dB background white noise. After 10 min, the mouse's startle response to a startle stimulus (110 or 120 dB white noise for 40 ms) was measured by a motion sensor for 140 ms. A test session was a random sequence of four trials each with a prepulse stimulus (74 or 78 dB white noise for 40 ms that preceded the startle stimulus by 100 ms) and two without. Six blocks of 6 trials were presented in pseudorandom order with the average inter-trial interval of 15 s.

Porsolt forced swim test
Each mouse was released in 7.5-cm-deep water at 23˚C in an acrylic cylinder (10 cm in diameter), and the duration of the motion for evacuation was measured up to 10 min automatically using ImagePS software (see Image analysis).

Monitoring social interaction and voluntary activity in the home cage
The position of each mouse housed alone in a cage was monitored from the top continuously for a week. The distance traveled along the diurnal cycle was measured automatically using ImageHA software (see Image analysis). Two mice of the same genotype that had been separately reared were housed together in a home cage and their 2D images from the top were captured at 1 fps for a week. Their physical contact and separation were represented respectively as 1 and 2 particles, and their locomotor activity was quantified by the differentials of pixels between successive frames by using ImageHA software (see Image analysis).

One-chamber social interaction test
The positions of two mice placed in a novel chamber (40 x 40 x 30 cm) were monitored from the top at 1 frame/sec. Their horizontal distance traveled and the number of contacts were measured automatically using ImageSI software (see Image analysis).

Crawley's three-chamber test for sociability and preference for social novelty
The apparatus had three chambers (20 × 40 × 22 cm) separated by two transparent partitions each with an opening (5 × 3 cm), and a lid with an infrared CCD camera. A male mouse (5-9 weeks old C57BL/6J, termed Stranger 1) that had no prior contact with the subject mice was enclosed in a cylinder cage (9 cm in diameter, set in the left chamber) that allowed nose contacts. Each subject mouse was released in the middle chamber and allowed to explore for 10 min, while the time spent in each chamber and within 5 cm from each cage was measured automatically using ImageCSI software (see Image analysis). Subsequently, another unfamiliar mouse (Stranger 2) was placed in another cylinder cage (in the right chamber) and monitored likewise for another 10 min.

Rota-rod test
Motor coordination and motor leaning were tested by measuring the survival duration on a 3-cm-thick rotating rod which was accelerated from 4 to 40 rpm over 5 min. Each mouse was subjected to 6 trials over 2 days.

Gait analysis
Automated gait analysis was conducted with DigiGait (Mouse Specifics Inc.). Each mouse was forced to walk on a transparent treadmill moving at 24 cm/sec, when the mouse body movement and paw footprints were captured at 150 frames/sec from underneath the treadmill. Multiple quantitative parameters (length, width and timing of the strides, paw angle etc.) were extracted from the time-lapse images with bundled software.

Hot plate test
Sensitivity and responses to a noxious stimulus were assessed by measuring the latency to the first response after placing a mouse on a metal plate at 55°C.

Tail suspension test
The movement of each mouse suspended by the tail at a height of 30 cm was recorded for 10 min and analyzed by using ImageTS software (see Image analysis).

Contextual and cued fear conditioning tests
Each mouse was exposed to a test chamber (26 x 34 x 33 cm) for 2 min, then to three pairs of a cue (55 dB white noise for 30 sec) each followed by a mild footshock (0.3 mA for 2 sec), repeated at 2-min intervals. For the context testing after 1 or 8 days, freezing was measured in the same chamber. For the cued testing in a distinct spatial context after 1 or 8 days, freezing after the noise was measured in a triangular chamber (35 x 35 x 41 cm) in a different room. The control of the stimuli, image acquisition at 1 frame/sec from the top, and image analysis were done automatically with ImageFZ software (see Image analysis). The criterion of freezing was defined when the difference of binarized mouse areas from two consecutive frames was below 10 pixels and lasted for 2 sec or longer.

Statistical analysis for behavioral tests
Quantitative data were expressed as mean ± SEM, and either one-way ANOVA or two-way repeated measures ANOVA was applied for statistical analyses unless otherwise noted.

Additional Figure Legends
Additional Figure  There was no difference in the sociability indices (S1 over E) between nTg and PrP-Sirt1 mice (n=19, 21). (D, E) In the social novelty preference test (Step2) after the sociability test (Step1), PrP-Sirt1 mice exhibited significantly less preference for S2 over S1 than nTg mice (p=0.038), indicative of their reduced curiosity in the novel social stimuli and/or augmented persistence to the previously-exposed social stimuli.
Additional Figure A12. The rotating rod (Rota-rod) test The duration in which nTg and PrP-Sirt1 mice (n=19, 21) kept pace with a rotating rod with a constant acceleration increased during 6 trials in two days. No statistically significant difference was observed in the motor coordination and motor learning.
Additional Figure A13. The gait analysis (A-I) Gait parameters extracted from high-speed digital images of nTg and PrP-Sirt1 mice on a treadmill (n=19, 21). No statistically significant difference was observed except for the wider hind step angle of PrP-Sirt1 mice (G).

Additional Figure A14. The hot plate test
Avoidance responses of nTg and PrP-Sirt1 mice (n=19, 21) to heat (55°C) given to the paws.
No statistically significant difference was observed.     Step 1