Physical activity delays hippocampal neurodegeneration and rescues memory deficits in an Alzheimer disease mouse model

The evidence for a protective role of physical activity on the risk and progression of Alzheimer's disease (AD) has been growing in the last years. Here we studied the influence of a prolonged physical and cognitive stimulation on neurodegeneration, with special emphasis on hippocampal neuron loss and associated behavioral impairment in the Tg4-42 mouse model of AD. Tg4-42 mice overexpress Aβ4-42 without any mutations, and develop an age-dependent hippocampal neuron loss associated with a severe memory decline. We demonstrate that long-term voluntary exercise diminishes CA1 neuron loss and completely rescues spatial memory deficits in different experimental settings. This was accompanied by changes in the gene expression profile of Tg4-42 mice. Deep sequencing analysis revealed an upregulation of chaperones involved in endoplasmatic reticulum protein processing, which might be intimately linked to the beneficial effects seen upon long-term exercise. We believe that we provide evidence for the first time that enhanced physical activity counteracts neuron loss and behavioral deficits in a transgenic AD mouse model. The present findings underscore the relevance of increased physical activity as a potential strategy in the prevention of dementia.


Open Field:
The open field test was used to asses both exploratory behavior and locomotor activity. The mice were tested using an open field box made of grey plastic with 50 x 50 cm surface area and 38 cm-high walls. Monitoring was done by an automated tracking system (AnyMaze, Stoelting). The percentage of time spent in the central part (20 x 20 cm) and the total active time was recorded (Jawhar et al., Neurobiol Aging 33: 196.e29-196.e40 (2012) mice. All data were given as means ± standard error of the mean (SEM) (***p < 0.001; *p < 0.05).

Supplementary Figure S3:
(a) Overview of voluntary wheel running performance of Tg4-42 hom mice in the recording period of 11 weeks. The average distance (km) per week is represented. (b) Exemplary picture of the tail hyperflexion phenotype due to the regular running wheel usage. All data were given as means ± standard error of the mean (SEM).
During the weeks of running wheel training mice were housed in single cages with food and water ad libitum. All animals had continuous voluntary access to a running wheel with a diameter of 11.3 cm placed inside the cage. A rotation sensor connected to the running wheel axis transmitted running activity with a resolution of 1/16 revolution and a sampling rate of 1/0.48 s to a customized recording device (Boenig und Kallenbach oHG, Dortmund, Germany). From this raw data, the average weekly running distance (km) was calculated and visualized using a custom-designed Matlab (The MathWorks, Inc., Natick, MA, USA) program. Animals were divided into two groups of equal size (n = 9). The running wheels of one group were freely movable whereas the running wheels of the other group were blocked, preventing the animals from running activity while providing the same enrichment of the environment.

Electrochemiluminescence Assay
Whole brain hemispheres were homogenized with 10 strokes of a glas-teflon homogenizer (800 rpm, CAT) in 700 µl lysis buffer (120 mM NaCl, 50 mM Tris, 1 % Triton X-100, 1 x Complete Mini-Protease Inhibitor, 1 x Complete Mini-Phosphatase inhibitor, dissolved in 10 ml ddH 2 O, pH 7.5) per 100 mg brain tissue. After centrifugation at 17000 x g at 4°C for 20 min, the supernatant containing the protein was stored at -80 °C until further use. Protein concentrations of lysates were determined using the Roti®-Quant universal kit according to the instructions of the supplier (Carl Roth). For determination of Aβ levels in whole brain hemispheres, an electrochemiluminescence total Aβ assay obtained from Meso Scale Discovery (Gaithersburg, MD, USA) was used. The Aβ assay is based on the Human (6E10) Abeta 40 Ultra-Sensitive kit. Here, the Aβ40 detection antibody is replaced by anti-Aβ 4G8 monoclonal antibody. Therefore, the total Aβ assay employs monoclonal antibody 6E10 (directed against an aminoterminal epitope of Aβ) for capture and the monoclonal antibody 4G8 (directed against Aβ17-26) for detection. As previously shown, 6E10 detects A4-42 equally well compared to full-length A1-42 or other N-truncated variants like A2-42 or A3-42 (Vanderstichele et al., Clin Chem 51(9): 1650-1660 (2005)). The assay was performed according to the protocol of the manufacturer and readout on a MSD QuickPLex SQ 120. In brief, a 96-well plate pre-coated with an Aβ antibody (6E10) was blocked with 3% BSA under shaking conditions at room temperature for 1 h. After 3 washing steps with 150 µl/well of 1X Tris Wash Buffer, 25µl of 2mg/ml protein lysates or calibrator was added per well and incubated under shaking conditions at room temperature for 1 h. After 3 additional washing steps, 25 µl of detection antibody solution (4G8) was added and again incubated with shaking for 1 h at room temperature in the dark. Upon 3 more washing steps, 150 µl of 1X Read Buffer T was added to each well and plate was read on MSD instrument.

Supplementary Figure S7:
Analysis of members of the HSP70 (HSPA1B) or HSP40 (DNAJA4) family revealed that these candidate genes are not per se altered in standard housed heterozygous Tg4-42 mice compared to standard housed WT mice. No significant differences in expression levels were detected (n = 6 per group).